Disease associated gene

ABSTRACT

The invention provides a disease-associated gene, the protein molecules encoded by the gene and polymorphic variants thereof, and the use of the gene and polymorphic variants thereof in diagnosis, prognosis and treatment of inflammatory or obstructive airways diseases.

FILED OF THE INVENTION

[0001] The present invention relates to a novel asthma-associated gene, designated AAGB, and to the protein molecule encoded by AAGB. The invention also relates to the use of AAGB polynucleotide sequences for diagnostic and prognostic screening of patient populations and the use of the protein encoded by AAGB as a therapeutic target.

BACKGROUND OF THE INVENTION

[0002] Asthma is a very common lung disease with the following characteristics: airways obstruction—this is usually reversible but often progressive chronic bronchial inflammation—a condition characterised by inflammatory cell infiltration and activation, release of biochemical mediators and structural changes (airway remodelling) bronchial hyperresponsiveness (BHR)—an exaggerated bronchoconstrictor response to a variety of immunologic, biochemical and physical stimuli.

[0003] Asthma is characterised clinically by chronic, intermittent airway obstruction with wheezing, coughing and breathlessness. Although asthma is typically associated with an obstructive impairment that is reversible, neither this finding nor any other single test or measure is adequate to diagnose asthma [Guidelines for the diagnosis and development of asthma, 1997, NIH Publication No. 97-4051]. Many diseases are associated with this pattern of abnormality. The patient's pattern of symptoms (along with other information from the patient's medical history) and exclusion of other possible diagnoses also are needed to establish a diagnosis of asthma. Clinical judgement is needed in conducting the assessment for asthma. Patients with asthma are heterogeneous and present signs and symptoms that vary widely from patient to patient as well as within each patient over time.

[0004] Many hypotheses have been advanced to explain the pathophysiology of asthma, including problems with airway smooth muscle, the role of inflammation, nervous innervation of the airways and mechanisms related to mediators. Although all of these factors may be important, it is unclear which are the primary (i.e. causative) defects and which are the secondary defects. It is generally agreed, however, that both the environment and genetics are important. Given the multifactorial nature of asthma, one approach to identifying the fundamental mechanisms is to discover asthma susceptibility genes that predispose individuals to develop asthma.

[0005] One method which can be used to identify asthma susceptibility genes is positional cloning. In this method, susceptibility genes are localised to a specific region of a human chromosome by using DNA markers to track the inheritance of the genes through families. DNA markers are fragments of DNA with a defined physical location on a chromosome, whose inheritance can be monitored. The closer a DNA marker is to a susceptibility gene, the greater the probability that the marker and the susceptibility gene will be passed together from parent to child. This phenomenon is called genetic linkage. Once linkage to a specific chromosomal region has been obtained, the size of the region is narrowed down using a combination of physical and genetic mapping until the region is small enough to be sequenced and the susceptibility gene can be identified. After identification of the susceptibility gene, any polymorphisms in this gene can be determined and an analysis performed to see whether these mutations occur with greater prevalence in asthmatics compared to non-asthmatics. The major advantages of positional cloning are that it is possible to identify novel genes even though the underlying factors causing the disease are unknown, and the genes identified are of direct pathological relevance (i.e. primary causative defects) because they make carriers directly susceptible to developing the disease.

[0006] In recent years a number of academic research groups have provided evidence for the presence of genes important in the regulation of asthmatic and allergic responses on human chromosome 5. In particular, evidence for the presence of susceptibility genes for BHR and elevated serum IgE levels on chromosome 5 in subregion 5q31-5q33 [Meyers et al., Genomics 23:464-470; Postma et al., N. Eng. J. Med. 333:894-900; and Bleecker et al., Clin. Exp. Allergy 25:84-88] was obtained from genetic linkage analysis of 92 Dutch asthma families. Strong evidence for genetic linkage between marker D5S436, raised total serum IgE levels [Meyers et al., Genomics 23: 464-470; Postma et al., N. Eng. J. Med. 333:894-900; and Bleecker et al., Clin. Exp. Allergy 25:84-88] and BHR [Postma et al., N. Eng. J. Med. 333:894-900; and Bleecker et al., Clin. Exp. Allergy 25:84-88] was found in the Dutch families.

[0007] No asthma susceptibility gene has yet been identified, so there is a need in the art for the identification of such genes. Identification of asthma susceptibility genes would provide a fundamental understanding of the disease process from which a number of clinically important applications would arise. Susceptibility genes identified may lead to the development of therapeutics (small molecule drugs, antisense molecules, antibody molecules) directly targeted to the gene or protein product of the gene, or may target the biochemical pathway of which the protein product is a part at an upstream or downstream location if the development of such drugs is easier than directly targeting the gene or its protein product. Polynucleotide sequences comprising the gene, sequence variants thereof and protein products thereof may be used to develop a clinical diagnostic test for asthma and for the identification of individuals at high risk for the development of asthma. The results of such tests may also have prognostic value and may be used to predict patients who respond to and those who do not respond to drug therapy. Finally, information about the DNA sequences of asthma susceptibility genes and the amino acid sequences encoded by these genes facilitates large scale production of proteins by recombinant techniques and identification of the tissues/cells naturally producing the proteins. Such sequence information also permits the preparation of antibody substances or other novel binding molecules specifically reactive with the proteins encoded by the susceptibility genes that may be used in modulating the natural ligand/antiligand binding reactions in which the proteins may be involved and for diagnostic purposes.

SUMMARY OF THE INVENTION

[0008] Accordingly, the present invention provides, in one aspect, an isolated polynucleotide, hereinafter alternatively referred to as AAGB, comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:4 or a functionally equivalent variant of said amino acid sequence, i.e. a variant thereof which retains the biological or other functional activity thereof, e.g. a variant which is capable of raising an antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:4.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows a graphic display of a region of chromosome 5.

[0010]FIG. 2 shows the results of Protean analysis of a putative protein.

[0011]FIG. 3 shows the genomic structure of the AAGB gene.

[0012]FIG. 4 shows the alignment of SEQ ID NO:4 with other protein sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Terms used herein have the following meanings:

[0014] “Isolated” refers to material removed from its original environment.

[0015] “Hybridization” or “hybridizes” refers to any process by which a strand of a polynucleotide binds with a complementary strand through base pairing.

[0016] “Stringent conditions” refer to experimental conditions which allow up to 20% base pair mismatches, typically two 15 minute washes in 0.1×SSC (15 mM NaCl, 1.5 mM sodium citrate, pH 7.0) at 65° C.

[0017] “Homology” or “homologous” refers to a degree of similarity between nucleotide or amino acid sequences, which may be partial or, when sequences are identical, complete.

[0018] “Expression vector” refers to a linear or circular DNA molecule which comprises a segment encoding a polypeptide of interest operably linked to additional segments which provide for its transcription.

[0019] “Antisense” refers to selective inhibition of protein synthesis through hybridisation of an oligo- or polynucleotide to its complementary sequence in messenger RNA (mRNA) of the target protein. The antisense concept was first proposed by Zamecnik and Stephenson (Proc. Natl. Acad. Sci. USA 75:280-284; Proc. Natl. Acad. Sci. USA 75:285-288) and has subsequently found broad application both as an experimental tool and as a means of generating putative therapeutic molecules (Alama, A., Pharmacol. Res. 36:171-178; Dean, N. M., Biochem. Soc. Trans. 24:623-629; Bennet, C. F., J. Pharmacol. Exp. Ther. 280:988-1000; Crooke, S. T., Antisense Research and Applications, Springer).

[0020] The term “variant” as used herein means, in relation to amino acid sequences, an amino acid sequence that is altered by one or more amino acids. The changes may involve amino acid substitution, deletion or insertion. In relation to nucleotide sequences, the term “variant” as used herein means a nucleotide sequence that is altered by one or more nucleotides; the changes may involve nucleotide substitution, deletion or insertion. A preferred functionally equivalent variant of the amino acid sequence SEQ ID NO:4 is one having at least 80%, more preferably at least 90%, and especially more than 95% amino acid sequence identity to SEQ ID NO:4.

[0021] By an amino acid sequence having x% identity to a reference sequence such SEQ ID NO:4, is meant a sequence which is identical to the reference sequence except that it may include up to 100-x amino acid alterations per each 100 amino acids of the reference sequence. For example, in a subject amino acid sequence having at least 80% identity to a reference sequence, up to 20% of the amino acid residues in the reference sequence may be substituted, deleted or inserted with another amino acid residue. Percentage identity between amino acid sequences can be determined conventionally using known computer programs, for example the FASTDB program based on the algorithm of Brutlag et al (Comp.App.Biosci. (1990) 6:237-245).

[0022] The isolated polynucleotide of the invention may be cDNA, genomic DNA or RNA. In particular embodiments, the isolated polynucleotide is cDNA comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5, a genomic DNA comprising the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO:7 or a DNA comprising a nucleotide sequence which hybridises to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 under stringent conditions.

[0023] The invention also provides an isolated polynucleotide comprising a consecutive 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair portion of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7.

[0024] A polynucleotide of the invention may be isolated by bioinformatics analysis of DNA sequences from the subregion 5q31-5q33 on chromosome 5 determined by sequencing of yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) and/or P1 artificial chromosomes (PACs) to identify genes within that subregion, searching for a sequence having greater than 95% identity to the predicted exon for a selected gene and isolating cDNA from a human fetal brain, adult brain, kidney, lung, muscle, peripheral blood lymphocyte, small intestine, spleen or testis cDNA library by PCR using primers designed using that sequence.

[0025] A polynucleotide of the invention, for example having the SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 may be prepared from the nucleotides which it comprises by chemical synthesis, e.g. automated solid phase synthesis using known procedures and apparatus.

[0026] In another aspect, the present invention provides an isolated polypeptide, particularly a recombinant polypeptide, comprising the amino acid sequence of SEQ ID NO:4 or a functionally equivalent variant thereof. Such a polypeptide may be produced by cloning a polynucleotide sequence as hereinbefore described into an expression vector containing a promoter and other appropriate regulating elements for transcription, transferring into prokaryotic or eukaryotic host cells such as bacterial, plant, insect, yeast, animal or human cells, and culturing the host cells containing the recombinant expression vector under suitable conditions. Techniques for such recombinant expression of polypeptides are well known and are described, for example, in J. Sambrook et al, Molecular Cloning, second edition, Cold Spring Harbor Press, 1990.

[0027] Accordingly, the present invention also provides a method of producing a polypeptide of the invention which comprises culturing a host cell containing an expression vector containing a polynucleotide sequence of the invention as hereinbefore described under conditions suitable for expression of the polypeptide and recovering the polypeptide from the host cell culture.

[0028] In another aspect, the present invention provides an expression vector containing a polynucleotide sequence of the invention as hereinbefore described.

[0029] The invention also provides an isolated polypeptide comprising a consecutive 10 amino acid portion identical in sequence to a consecutive 10 amino acid portion of SEQ ID NO:4.

[0030] A polypeptide of the invention may be expressed as a recombinant fusion protein with one or more heterologous polypeptides, for example to facilitate purification. For example, it may be expressed as a recombinant fusion protein with a heterologous polypeptide such as a polyhistidine containing a cleavage site located between the polynucleotide sequence of the invention and the heterologous polypeptide sequence, so that the polypeptide comprising the amino acid sequence of SEQ ID NO:4 may be cleaved and purified away from the heterologous moiety using well known techniques.

[0031] A polypeptide of the invention may also be synthesised, in whole or in part, from the amino acids which it comprises using well known chemical methods, for example automated solid phase techniques.

[0032] Isolated polypeptides of the invention as hereinbefore described may be purified by well known standard procedures.

[0033] The present invention also provides a variant of a polynucleotide of the invention as hereinbefore described, particularly a polynucleotide having a SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7, which contains a sequence polymorphism correlated with a disease, particularly asthma. The polymorphism may be an addition, deletion or replacement of one or more nucleotides. Single nucleotide polymorphisms (SNPs), as compared with SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7, which have been identified in genetic samples from asthmatic patients are shown hereinafter in the Examples.

[0034] The present invention further provides a variant of the polypeptide comprising the amino acid sequence of SEQ ID NO:4, which variant (hereinafter described alternatively as mutant protein) is an isolated polypeptide which is encoded by a variant of a nucleotide sequence of a polynucleotide of the invention as hereinbefore described, particularly a variant of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7, having a sequence polymorphism correlated with a disease, particularly asthma. Such mutant proteins can be expressed or synthesised analogously to the polypeptide comprising SEQ ID NO:4. The present invention also provides an antibody which is immunoreactive with a polypeptide of the invention as hereinbefore described, or a mutant protein as hereinbefore described. The antibody may be a polyclonal or monoclonal antibody. Such antibodies may be prepared using conventional procedures. Methods for the production of polyclonal antibodies against purified antigen are well established (cf. Cooper and Paterson in Current Protocols in Molecular Biology, Ausubel et al. Eds., John Wiley and Sons Inc., Chapter 11). Typically, a host animal, such as a rabbit, or a mouse, is immunised with a purified polypeptide or mutant protein of the invention, or immunogenic portion thereof, as antigen and, following an appropriate time interval, the host serum is collected and tested for antibodies specific against the polypeptide. Methods for the production of monoclonal antibodies against purified antigen are well established (cf. Chapter 11, Current Protocols in Molecular Biology, Ausubel et al. Eds., John Wiley and Sons Inc.). For the production of a polyclonal antibody, the serum can be treated with saturated ammonium sulphate or DEAE Sephadex. For the production of a monoclonal antibody, the spleen or lymphocytes of the immunised animal are removed and immortalised or used to produce hybridomas by known methods. Antibodies secreted by the immortalised cells are screened to determine the clones which secrete antibodies of the desired specificity, for example using Western blot analysis. Humanised antibodies can be prepared by conventional procedures.

[0035] In another aspect, the present invention provides an antisense oligonucleotide comprising a nucleotide sequence complementary to that of a polynucleotide of the invention or a variant thereof having a polymorphism correlated with a disease, particularly asthma, in particular a nucleotide sequence complementary to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7, or a polymorphic variant thereof, The antisense oligonucleotide may be DNA, an analogue of DNA such as a phosphorothioate or methylphosphonate analogue of DNA, RNA, an analogue of RNA, or a peptide nucleic acid (PNA). The antisense oligonucleotides may be synthesised by conventional methods, for example using automated solid phase techniques.

[0036] The present invention also provides a polynucleotide probe comprising at least 15 contiguous nucleotides of a polynucleotide of the invention or a complement thereof. The probe may be cDNA, genomic DNA or RNA. Usually it is a synthetic oligonucleotide comprising 15 to 50 nucleotides, which can be labelled, e.g. with a fluorophore, to provide a detectable signal.

[0037] The polynucleotide probe is capable of selectively hybridising under stringent conditions to a polynucleotide fragment having a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5, 6 and 7. The probe has a sequence such that under such hybridisation conditions it hybridizes only to its cognate sequence. DNA probes as described above are useful in a number of screening applications including Northern and Southern blot analyses, dot blot and slot blot analyses, and fluorescence in situ hybridisation (FISH).

[0038] The present invention also includes a pair of oligonucleotides having nucleotide sequences useful as primers for DNA amplification of a fragment of a polynucleotide of the invention, i.e. of the human AAGB gene (hAAGB), wherein each primer of said pair is at least 15 nucleotides in length and said pair have sequences such that when used in a polymerase chain reaction (PCR) with either human genomic DNA or a suitable human cDNA target they result in synthesis of a DNA fragment containing all or preferably part of the sequence of hAAGB. The primer pair is preferably capable of amplifying at least one exon of hAAGB (or portion thereof), such as an exon selected from those in SEQ ID NO:1, 2, 3, 5, 6 or 7. Examples of such primer pairs are shown hereinafter in the Examples. Exemplary applications of such primer pairs include amplification of DNA fragments for use in the detection of changes to the polynucleotide sequence in asthmatic patients as shown hereinafter in the Examples.

[0039] The role of the polypeptide of the invention in asthma and other obstructive or inflammatory airways diseases characterised by bronchial hyperresponsiveness can be determined using conventional allergen driven animal models for bronchial hyperresponsiveness, e.g. the ovalbumin-induced BHR mouse model (Tsuyuki et al, J. Clin. Invest. 96:2924-2931) or the guinea pig model hereinafter described.

[0040] Polynucleotides, polypeptides, antibodies, antisense oligonucleotides or probes of the invention as hereinbefore described, hereinafter alternatively referred to collectively as agents of the invention, may be used in the treatment (prophylactic or symptomatic) or diagnosis of inflammatory or obstructive airways diseases. For example, a polypeptide of the invention may be used to treat a mammal, particularly a human, deficient in or otherwise in need of that polypeptide; a polynucleotide of the invention may be used in gene therapy where it is desired to increase AAGB activity, for instance where a subject has a mutated or missing AAGB gene; an antisense oligonucleotide of the invention may be used to inhibit AAGB activity or activity of variants of the AAGB gene having a polymorphism correlated with a disease, e.g. asthma, where this is desired; an antibody of the invention may be used to detect, or determine the level of expression of, AAGB polypeptides or a disease-correlated polymorphic variant thereof, or to inhibit ligand/antiligand binding activities of AAGB polypeptides; and a probe of the invention may be used to detect the presence or absence of the AAGB gene, i.e. to detect genetic abnormality.

[0041] “Gene therapy” refers to an approach to the treatment of human disease based upon the transfer of genetic material into somatic cells of an individual. Gene transfer can be achieved directly in vivo by administration of gene-bearing viral or non-viral vectors into blood or tissues, or indirectly ex vivo through the introduction of genetic material into cells manipulated in the laboratory followed by delivery of the gene-containing cells back to the individual. By altering the genetic material within a cell, gene therapy may correct underlying disease pathophysiology. Suitable vectors, and procedures, for gene delivery to specific tissues and organ systems in animals are described in Dracopoli, N. C. et al., Current Protocols in Human Genetics. John Wiley and Sons Inc., Chapters 12 and 13 respectively. In relation to polynucleotides of the invention, gene therapy may involve delivery of a viral or non-viral gene therapy vector containing an expression cassette of the AAGB gene under suitable control elements to the lungs of diseased individuals (eg. asthmatics) so that the underlying disease pathophysiology is corrected or ameliorated.

[0042] Accordingly, in further aspects, the present invention provides

[0043] a pharmaceutical composition comprising a polynucleotide, polypeptide, antibody or antisense oligonucleotide of the invention as hereinbefore described, optionally together with a pharmaceutically acceptable carrier;

[0044] a method of treating an inflammatory or obstructive airways disease which comprises administering to a subject in need thereof an effective amount of a polynucleotide, polypeptide, antibody or antisense oligonucleotide of the invention as hereinbefore described;

[0045] a method of detecting genetic abnormality in a subject which comprises incubating a genetic sample from the subject with a polynucleotide probe of the invention as hereinbefore defined, under conditions where the probe hybridises to complementary polynucleotide sequence, to produce a first reaction product, and comparing the first reaction product to a control reaction product obtained with a normal genetic sample, where a difference between the first reaction product and the control reaction product indicates a genetic abnormality in the subject or a predisposition to developing a disease such as asthma;

[0046] a method of detecting the presence of a polynucleotide of the invention, e.g. comprising SEQ ID NO:1, 2, 3, 5, 6 or 7, in cells or tissues which comprises contacting DNA from the cell or tissue with a polynucleotide probe as hereinbefore defined under conditions where the probe is specifically hybridizable with a polynucleotide of the invention, and detecting whether hybridization occurs;

[0047] a method of detecting an abnormality in the nucleotide sequence of a polynucleotide of the invention in a patient which comprises amplifying a target nucleotide sequence in DNA isolated from the patient by a polymerase chain reaction using a pair of primers as hereinbefore described which target the sequence to be amplified and analysing the amplified sequence to determine any polymorphism present therein; and

[0048] a method of detecting polymorphism in a subject which comprises treating a tissue sample from the subject with an antibody to a mutant protein of the invention and detecting binding of said antibody.

[0049] The term “polymorphism” means any sequence difference as compared with the sequence of a polynucleotide of the invention as hereinbefore described.

[0050] Hybridisation of a polynucleotide probe of the invention with complementary polynucleotide sequence may be detected using in situ (eg. FISH) hybridization, Northern or Southern blot analyses, dot blot or slot blot analyses. The abnormality may also be detected for example by conformation sensitive gel electrophoresis (CSGE) and DNA sequencing as described hereinafter in the Examples. The genetic abnormality may result in a change in the amino acid sequence of the individual's AAGB protein relative to the the amino acid sequence of a normal hAAGB protein, or loss of protein. Alternatively, the change may not alter the amino acid sequence but may instead alter expression of the AAGB gene by altering the sequence of controlling elements either at the 5′-, or 3′-end of the gene, or altering the sequence of control elements within intronic regions of the gene. Changes may also affect the way the gene transcript is processed or translated. The invention also includes kits for the detection of an abnormality in the polynucleotide sequence of an individual's AAGB gene. Hybridisation kits for such detection comprise a probe of the invention as hereinbefore described, which probe may be modified by incorporation of a detectable, e.g. chemiluminescent or fluorescent, label therein, and may include other reagents such as labelling reagents, i.e. reagents to incorporate a detectable label such as a radioactive isotope, chemiluminescent or fluorescent group into a hybridised product, and buffers. PCR amplification kits comprise primer pairs such as those described above together with a DNA polymerase such as Taq polymerase, and may include additional reagents, such as an amplification buffer and the like. Specific embodiments of the PCR amplification kits can include additional reagents specific for a number of techniques that detect polynucleotide changes, including CSGE and DNA sequencing.

[0051] The effectiveness of an agent of the invention in inhibiting or reversing airways hyperreactivity may be demonstrated in a guinea pig test model. The acute injection of pre-formed immune complex renders guinea pigs hyperreactive to histamine. Doses of histamine which cause only a small degree of bronchoconstriction prior to administration of immune complex cause a much stronger effect thereafter. Guinea-pigs (Dunkin-Hartley, male, 400-600 g) are anaesthetised with phenobarbital (100 mg/kg i.p.) and pentobarbital (30 mg/kg i.p.) and paralysed with gallamine (10 mg/kg i.m.) and ventilated with a mixture of air and oxygen (45:55), v/v). Animals are ventilated (8 ml/kg, 1 Hz) via a tracheal cannula. Ventilation is monitored by a flow transducer. When making measurements of flow, coincident pressure changes in the thorax are monitored directly via an intrathoracic trochar, permitting display of differential pressure relative to the trachea. From this information resistance and compliance are calculated at each inspiration. An allergic reaction is initiated by intravenous injection of preformed immune complexes (prepared by adding 30 μg of bovine gamma globulin in 0.05 ml of saline to 0.05 ml of guinea pig anti-bovine gamma globulin anti-serum) 3 times at 10 minute intervals. Intravenous injections of histamine (1.0-3.2 μg/kg at 10 minute intervals) are used to define the sensitivity of the airways prior to and following the last exposure to the immune complex. Airways hyperreactivity is expressed as the paired difference for the maximal value of lung resistance in response to histamine before and after repeated injection of immune-complex. The agents of the invention are administered intratracheally either as solutions or suspensions in tragacanth. The ED₅₀₋ values for reversal of airways hyperreactivity are determined graphically from the dose response curves and represent those doses which cause a 50% reduction of airways hyperreactivity.

[0052] Inflammatory or obstructive airways diseases to which the present invention is applicable include asthma of whatever type or genesis including both intrinsic (non-allergic) asthma and extrinsic (allergic) asthma. Treatment of asthma is also to be understood as embracing treatment of subjects, e.g. of less than 4 or 5 years of age, exhibiting wheezing symptoms and diagnosed or diagnosable as “wheezy infants”, an established patient category of major medical concern and now often identified as incipient or early-phase asthmatics. (For convenience this particular asthmatic condition is referred to as “wheezy-infant syndrome”.)

[0053] Prophylactic efficacy in the treatment of asthma will be evidenced by reduced frequency or severity of symptomatic attack, e.g. of acute asthmatic or bronchoconstrictor attack, improvement in lung function or reduced airways hyperreactivity. It may further be evidenced by reduced requirement for other, symptomatic therapy, i.e. therapy for or intended to restrict or abort symptomatic attack when it occurs, for example anti-inflammatory (e.g. corticosteroid) or bronchodilatory. Prophylactic benefit in asthma may in particular be apparent in subjects prone to “morning dipping”. “Morning dipping” is a recognised asthmatic syndrome, common to a substantial percentage of asthmatics and characterised by asthma attack, e.g. between the hours of about 4 to 6 am, i.e. at a time normally substantially distant form any previously administered symptomatic asthma therapy.

[0054] Other inflammatory or obstructive airways diseases and conditions to which the present invention is applicable include adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary or airways disease (COPD or COAD), including chronic bronchitis, or dyspnea associated therewith, emphysema, as well as exacerbation of airways hyperreactivity consequent to other drug therapy, in particular other inhaled drug therapy. The invention is also applicable to the treatment of bronchitis of whatever type or genesis including, e.g., acute, arachidic, catarrhal, croupus, chronic or phthinoid bronchitis. Further inflammatory or obstructive airways diseases to which the present invention is applicable include pneumoconiosis (an inflammatory, commonly occupational, disease of the lungs, frequently accompanied by airways obstruction, whether chronic or acute, and occasioned by repeated inhalation of dusts) of whatever type or genesis, including, for example, aluminosis, anthracosis, asbestosis, chalicosis, ptilosis, siderosis, silicosis, tabacosis and byssinosis.

[0055] Having regard to their anti-inflammatory activity, in particular in relation to inhibition of eosinophil activation, agents of the invention are also useful in the treatment of eosinophil related disorders, e.g. eosinophilia, in particular eosinophil related disorders of the airways (e.g. involving morbid eosinophilic infiltration of pulmonary tissues) including hypereosinophilia as it effects the airways and/or lungs as well as, for example, eosinophil-related disorders of the airways consequential or concomitant to Löffler's syndrome, eosinophilic pneumonia, parasitic (in particular metazoan) infestation (including tropical eosinophilia), bronchopulmonary aspergillosis, polyarteritis nodosa (including Churg-Strauss syndrome), eosinophilic granuloma and eosinophil-related disorders affecting the airways occasioned by drug-reaction.

[0056] The agents of the invention may be administered by any appropriate route, e.g. orally, for example in the form of a tablet or capsule; parenterally, for example intravenously; topically, e.g. in an ointment or cream; transdermally, e.g. in a patch; by inhalation; or intranasally.

[0057] Pharmaceutical compositions containing agents of the invention may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus oral dosage forms may include tablets and capsules, and compositions for inhalation may comprise aerosol or other atomizable formulations or dry powder formulations.

[0058] The invention includes (A) an agent of the invention in inhalable form, e.g. in an aerosol or other atomizable composition or in inhalable particulate, e.g. micronised form, (B) an inhalable medicament comprising an agent of the invention in inhalable form; (C) a pharmaceutical product comprising such an agent of the invention in inhalable form in association with an inhalation device; and (D) an inhalation device containing an agent of the invention in inhalable form.

[0059] Dosages of agents of the invention employed in practising the present invention will of course vary depending, for example, on the particular condition to be treated, the effect desired and the mode of administration. In general, suitable daily dosages for administration by inhalation are of the order of 1 μg to 10 mg/kg while for oral administration suitable daily doses are of the order of 0.1 mg to 1000 mg/kg.

[0060] The present invention further provides a method of determining predisposition of a subject to asthma comprising determining the presence or absence in DNA from the subject of a sequence polymorphism in a polynucleotide of the invention which correlates with asthma.

[0061] In another aspect, the present invention provides a method of determining predisposition of a patient to asthma which comprises identifying in DNA from the patient a sequence polymorphism or haplotype in a polynucleotide of the invention, as compared with a normal control DNA from a non-asthmatic subject, which correlates with asthma. A haplotype is a set of polymorphisms which is inherited together as a group.

[0062] In a related aspect, the invention provides a method of determining predisposition of a patient to asthma which comprises identifying in DNA from the patient a sequence polymorphism or haplotype in a polynucleotide of the invention, as compared with SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7, which correlates with asthma.

[0063] Identification of a sequence polymorphism may be effected by conventional sequencing and sequence analysis techniques, for example as described in Cotton, R. G. H., Mutation Detection, Oxford University Press, 1997; Landegren, U., Laboratory Protocols for Mutation Detection, Oxford University Press; and R. G. H. Cotton et al, Mutation Detection, Oxford University Press, 1998.

[0064] Sequence polymorphisms which correlate with asthma may alter the amino acid sequence in the encoded polypeptide or may affect expression levels of the polypeptide or the way in which a transcript is processed.

[0065] Certain sequence polymorphisms or haplotypes may correlate with the severity and/or nature of the asthmatic phenotype, e.g. with mild, moderate or severe asthma as defined by established clinical parameters. Identification of polymorphisms may therefore be useful for prognosis, determination of therapeutic strategy and prediction of patient responses to therapy.

[0066] A polypeptide or mutant protein of the invention can be used to identify enhancers (agonists) or inhibitors (antagonists) of its activity, i.e. to identify compounds useful in the treatment of inflammatory or obstructive airways diseases, particularly asthma. Accordingly, the invention also provides a method of identifying a substance which modulates the activity of a polypeptide of the invention comprising combining a candidate substance with a polypeptide or mutant protein of the invention and measuring the effect of the candidate substance on said activity. The activity of a polypeptide or mutant protein of the invention may be measured, for example, by measuring changes in levels of intracellular components such as Ca²⁺ or cAMP, by a shape change assay or by an appropriate reporter gene assay. The invention also includes a method of identifying a substance which binds to a polypeptide or mutant protein of the invention comprising mixing a candidate substance with a polypeptide or mutant protein of the invention and determining whether binding has occurred.

[0067] The invention is illustrated by the following Examples, with reference to the accompanying drawings.

[0068] Abbreviations used in the Examples have the following meanings: AEBSF: 4-(2-Aminoethyl)benzenesulfonyl fluoride BAC: bacterial artificial chromosome BAP: 1,4-bis(acryloyl)piperazineBLAST: basic local alignment search tool BSA: bovine serum albumin CSGE: conformation sensitive gel electrophoresis DNTP deoxynucleotide triphosphate DTT: dithiothreitol EGF: epidermal growth factor EIA: enzyme immunoassay EST: expressed sequence tag FCS: fetal calf serum GPCR: G-protein coupled receptor MTN: multiple tissue northern NCBI: National Center for Biotechnology Information ORF: open reading frame PAC: P1 artificial chromosome PBS: phosphate buffered saline PBL: peripheral blood lymphocyte PEG: polyethylene glycol PMSF: Phenylmethylsulfonyl fluoride SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis SNP single nucleotide polymorphism STS sequence tagged site TGF: transforming growth factor TTE: 44 mM Tris, 14.5 mM taurine, 0.1 mM EDTA, pH 9.0

EXAMPLE 1

[0069] Bacterial artificial chromosome (BAC) clones identified using physical map information for human chromosome 5q31-q33 publicly available on the Lawrence Berkley National Laboratory Genome Centre web site (LBNL; www-hgc.lbl.gov/biology/bacmap/2.gif) obtained as BAC clone numbers h164 (22f14), c5 (50g20), h187 (35k5), h167 (8e5) and h177 (32d16) from Research Genetics (Huntsville, Ala., USA), and a P1 artificial chromosome (PAC) isolated by PCR using primers with SEQ ID NOS:11 to 14 for the STS markers bac51107T (5′ end of BAC 50g20) and bac51330T (3′ end of BAC 22f14) available on the LBNL website (www₁₃hgc.lbl.gov/sts.html) by Genome Systems Inc. (St. Louis, Mo., USA), the BACs and PAC together covering a sub-region of human chromosonal region 5q31-5q33, are sequenced using conventional techniques for an ABI 377 sequence (http://www.pebio.com/ab/about/dna/377/). The resulting genomic DNA sequence is analysed using SCAN (Burge and Karlin, J. Mol. Biol. 268:78-94) and GENEMARK version 2.4 (Borodovsky and McIninch, Comp. Chem. 17:123-133) gene-finding programs and BLAST (Altschul et al., J. Mol. Biol. 215:403-410) homology searches against public protein, EST and DNA databases (SWISSPROT, SWISSPROTPLUS, GenBank, Genbank updates, EMBL, GENEMBLPLUS, GenBank EST, EMBL EST, GenBank STS, EMBL STS), the results of which are parsed into a human chromosome 5-specific version of ACeDb (A C. elegans Database; http://www.sanger.ac.uk/Software/Acedb/) for graphic display. From this graphic display, shown in FIG. 1, significant regions (i.e. genes) are identified by predicted exons and alignedEST/protein hits. A gene AAGB is initially identified on the graphic display as a GENSCAN-predicted gene covering 55 kb of genomic DNA and comprising 9 exons ranging in size from 31-132 bp: GENSCAN- Nucleotide Position In: Exon Size Predicted Exon SEQ ID No. 6 SEQ ID No. 7 (bp) 1 1212-1274 — 63 2 1343-1474 — 132 3 24053-24134 — 82 4 24451-24581 — 131 5 27969-28056 — 88 6 29972-30096 — 125 7 32801-32867 — 67 8 — 6209-6239 31 9 — 6486-6564 79

[0070] A number of hits are found in the EST database. From these EST hits a Unigene cluster (Hs.9788) is identified. Assembly of the ESTs in this cluster using the SeqMan module of LAsergene software (DNASTAR, Inc., Madison, Wis., USA) produces 2 contigs of ESTs of 2057 bp (SEQ ID NO:1) and 725 bp (SEQ ID NO:2). PCR primers having SEQ ID NOS: 8, 9 and 10 were used to screen cDNA libraries from fetal brain, adult brain, kidney, lung, muscle, PBL, small intesrine, spleen and testis. All give a positive result on screening, but the longest clone is found in the small intestine library. The cDNA insert is sequenced using primer-directed walking. The resulting 1054 base pair of insert sequence (SEQ ID NO:5) aligns with sequence SEQ ID NO:1 at position 859-1893, therefore, the cDNA sequence of SEQ ID NO:5 does not extend the Unigene cluster sequence, or bridge the sequence gap between SEQ ID NO:1 and SEQ ID NO:2. Using the reverse complement of SEQ ID NO:2 positions 725-605, a 3599 bp EST clone (232376.4; SEQ ID NO:3) is identified by BLAST screening of the LifeSeq® Gold commerical database (Incyte Pharmaceuticals Inc., 3160 Porter Drive, Palo Alto, Calif. 94304, USA). Alignment of SEQ ID NOS:1, 2 and 3 shows that clone 232376.4 contains the DNA sequences represented by SEQ ID NOS:1 and 2 and that the DNA sequence between positions 1942 and 2887 of SEQ ID NO:3 bridges the 946 bp gap between SEQ ID NOS: 1 and 2. Alignment of SEQ ID NO: 3 with the genomic sequence (SEQ ID NO:6) reveals 8 exons spread over 45 kb of genomic DNA. The genomic structure of the AAGB gene and its to SEQ ID NOS: 1, 2, 3, 5 and 6 are shown in FIG. 3. Nucleotide Position Exon SEQ ID NO: 3 in SEQ ID NO: 6 Exon Size (bp) 1  48-285* 1037-1274 238 2 286-373* 24047-24132 88 3 374-505* 24451-24582 132 4 506-592* 27970-28056 87 5 593-717* 29972-30096 125 6 718-785* 32801-32868 68 7 786-890  36815-36920 106 8 891-3599 43976-46684 2708

[0071] Analysis of SEQ ID NO:3 using the EditSeq module of the Lasergene software (DNASTAR, Inc., Madison, Wis., USA) reveals that the longest open reading frame (ORF) encodes a protein of 221 amino acids (SEQ ID NO:4; encoded by nucleotides 223-888 of SEQ ID NO: 3). Analysis of this putative protein using the Kyte-Doolittle algorithm (Kyte and Doolittle J. Mol. Biol. 157:105-132) in the Protean module of the Lasergene software indicates the presence of 4 hydrophilic domains in the N-terminal half of the molecule and 3 hydrophobic domains in the C-terminal half, indicating that the protein is a transmembrane protein (FIG. 2).

[0072] Further analysis on the SOSUI system (http://azusa.proteome.bio.tuat.ac.jp/sosui/) identifies 3 transmembrane helices corresponding to the 3 predicted hydrophobic domains: N C No. Terminal Transmembrane Region Terminal Length 1 117 IFMLTFFMAFLFNWIGFFLSFCL 139 23 2 148 GAISGFGLSLIKWILIVRFSTYF 170 23 3 179 WLWWVFLVLGFLLFLRGFINYAK 201 23

[0073] Further analysis of the three transmembrane domains by searches against the MOTIF database (www.motif.genome.ad.jp) reveals hits for all three domains against the rhodopsin-like GPCR superfamily signature [Attwood and Findlay, Prot. Engng. 7:195-203 and 6: 167-176]. The presence of only three hydrophobic membrane-spanning regions, rather than seven (typical signature of a GPCR), indicates that the protein encoded by 232376.4 is not a member of the GPCR family, but is a member of an alternative family of transmembrane proteins.

[0074] BLASTP searching of the NR protein database using SEQ ID NO:4 as the query sequence identifies significant hits with protein sequences AK001723 (putative protein translation of a human cDNA derived from a teratocarcinoma cell line), AB032991 (KIAA1165 protein derived from a cDNA clone derived from an adult male brain cDNA library) and AAF49316 (CG8056 gene product; a predicted protein from the Drosophila sequencing effort). Alignment of SEQ ID NO: 4 with the AK001723, AB032991 and AAF49316 protein sequences using Clustal 1.7 (Thompson, Higgins and Gibson Nucleic Acids Res. 22:4673-4680) shows that the three predicted transmembrane domains in 232376.4 are conserved in the three protein database hits (FIG. 4).

[0075] Moreover, the alignment shows that AB032991 and AK001273 are identical, except that the former is 40 amino acids longer. The gene encoding AB032991/KIAA1165 is expressed in a number of tissues and has been assigned to chromosome 13 by radiation hybrid mapping (http://www.kasuza.or.jp/huge). Examination of the FlyBase (http://flybase.bio.indiana.edu:7081/) database for further details of the AAF49316/CG8056 protein (Fban0008056) reveals that the protein is encoded by the Drosophila Keren gene. The Keren gene encodes two putative proteins of 217 amino acids (alt1; AAF49315) and 267 amino acids (alt 2; AAF49316). Alt 1 and alt 2 do not display any homology with each other and only alt 2 has homology with SEQ ID NO: 4. All protein sequences similar to alt 1 and alt 2 in the NR database are accessed using the Drosophila precomputed BLAST facility at the NCBI (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/7227.html). Alt 1 has homologies with 223 proteins in a range of organisms, many of which are transmembrane proteins containing EGF motifs. In contrast, alt 2 has a proline rich motif and displays homologies to only 2 other proteins: AB032991/KIAA1165 and AAF11803 (a putative erythomycin esterase from Deinococcus radiodurans).

[0076] Taken together, the Protean, SOSUI and BLAST analyses indicate that the AAGB gene encodes a novel transmembrane protein.

[0077] Using the cDNA clone insert corresponding to SEQ ID NO:5, a northern blot of mRNA from a number of human tissues (human 12-lane MTN blot; Clontech Laboratories UK Ltd., Basingstoke, Hampshire, UK) is probed to examine the expression profile of gene AAGB. Two hybridising bands of ˜4.0 kb and 2.0 kb are detected which are most strongly visulaised in brain, heart, kidney and liver RNA and at lower levels in skeletal muscle, spleen, placenta and lung. Very low levels are found in colon, thymus, small intestine and peripheral blood lympocytes. PCR analysis of first-strand cDNAs derived from 24 different tissues (Rapid-Scan™ Gene Expression Panel; OriGene Technologies Inc., Rockville, Md., USA) using primers having SEQ ID NOS:15 and 18 confirms the Northern blot results.

EXAMPLE 2

[0078] In this example conformation sensitive gel electrophoresis (CSGE: Ganguly et al., Proc. Natl. Acad. Sci. USA 90:10325-10329; Ganguly and Williams, Hum. Mut. 9:339-343) is used to detect potential sequence changes in PCR-amplified DNA fragments from blood DNA isolated from asthmatic patients. Single base mismatches in DNA heteroduplexes are detected by polyacrylamide gel electrophoresis in the presence of mildly denaturing solvents which amplify the tendency of mismatches to produce conformational changes and result in differential migration of homo-duplexes and heteroduplexes. To generate heteroduplexes, amplified PCR products are thermally denatured, annealed, then analysed by polyacrylamide gel electrophoresis. DNA fragments are visualised by ethidium bromide staining. DNA fragments showing differential electrophoretic migration patterns are then sequenced to confirm the presence of a change to the polynucleotide sequence and the exact nature of this change.

[0079] PCR primer sets covering the 8 exons (including the exon-intron boundaries) identified by alignment of SEQ ID NO:3 with SEQ ID NO: 6 (Example 1), the first 1 kb of intron 1 and 1 kb of the 3′-end of the AAGB gene are designed using SEQ ID NO:6 and Primer Express™ (version 1.0; Perkin Elmer, P/N 604313). These primer sets (SEQ ID NOs: 18-84) are: Alias Forward Reverse Prom 1.1 CTTCACCCCYIAGTAGCTCTGTGTC AATCCTTGAGAACTGCTTGGATCT Prom 1.2 CCCTTTGCACCTGTTCCTGT TCCGGTCTGGCACCATG Prom 1.3 AAATGTCAGATAGCAAGTATTATAGGGTTTG TGCCAAGCACTGAGCTTCAC Prom 1.4 AACCACGATTCTGCTGCCA GCCCCACCCGCGACT Prom 1.5 ACACTTGAGCCTCGGAGGC CAGGGAATCAGAGCGAGCG Exon 1   CCCGCCTCTTCCCCAG AGCCCCGGCGTCGA Intron 1.1 TGTCGACTGGGCCATGG CCGCCTGTGGGTCGC Intron 1.2 GCCTCAGGCCTCTCTGGC GAGCTATGGGCAGCAGCAG Intron 1.3 TCCCTGAGTCAGAACCCGC AATACACCCGGCACACGC Intron 1.4 CCCCTCGGCTCCGTTTT CCACCATTACTGTATGTTAGATTAACACTTC Intron 1.5 TCATTTTCTCGGGTTTGATTTAATACA AAACCAGTAACCGGCCG Exon 2   TCCATAGAATAACTTCCTGGGAGTAGAAT TTGTAACCTTTCTTCCAACTGCTTC Exon 3   TGGTTCAATTGTTATGTATGTACTCTTAAGG AAAATTAGCTCTACTTTCACTAATCAATACAAT Exon 4   GAATTTTAAACACACATTGAGCCTTTAAT CCAGTAAAAGGCACCAAATAAACATA Exon 5   GGCAGGTGTAAGGCACTCTTG TGTGCATGAACCATTTGCAA Exon 6   GCATTTTGTGATTTTGTGTACCCTT TTAGGAAGAACCTACAACCACAAAGA Exon 7   CAGATTCTGGAGGAAATTGCTAATGT GGGACTGTGCCTTCTTTACTTTCTCT Exon 8.a TTGTCTTAGCCAAAATAACAAGCTAACTAGA GGACATGGCTCATGCTACTTAAAATG Exon 8.b GATCATTTCTCTCTATTTGTTCCTAGGTGT CATACAGAATTGAATAAATTTCTAGATTAACTTGAA Exon 8.c CATGTCCCTGTAGTCGGTAGGG GTTTTATACAGTGACAACACCTTTAGAATCC Exon 8.d AACACCTGGAAGCAAAATCATAGTG GGATCATCTCAAAAGTTCCAAGCTACT Exon 8.e AATTCTTGACACTCTTTCTATAATTAGCGTTC ATCAACAAATGCAAGCCAAGAAAT Exon 8.f AATGTGTTAGCAGAAACCAGTGGG CTCTACAACTCCTCTCTACTTTTTTAGTCCAT Exon 8.g TTGTATGGCCTATAATGTTCTAAGTTCCA CCACTTCATGTGCCTCAAAAGATT Exon 8.h CGCCTTATTTGGTTTTGTATATTCAAC CGCGGTTCCTATGAATACCTTTC Exon 8.i GAGTCCTTACTGGTATTCAGGATGAAA CAAGCAAATTTTAATAATGCCTTTTATTTC Exon 8.j GTTATGGAGTAAACTAGCTTGGACCTTG TTCATCTGTACTAATAATGACTCACGGTG 3′.1 GCTGGAATGCAGTGTTGCG TTGAAATATCACTTCTATTGGTCCTGA 3′.2 GACCTCGTGATCCGCCC ACAAGAAGCACGGTCAAATGATT 3′.3 AATTTGAGTGATTGCCTGAGGG GCCAAGATCACGACTTTGTACTTAAGT 3′.4 ACAAAGTCGTGATCTTGGCTCA GCTGAACCACCCACAGAACCT 3′.5 GTTGTGACCCCTATTACCTATGTGAAT CACAAACGTGTACAGGTATACGCAA 3′.6 GGTGGATTGGGTGAGAGCATT CTATAGGCCATAAAGTTGTAGGTGATACA

[0080] Using the above primer sets, 33 polynucleotides are amplified from blood DNA samples from 16 asthmatic patients. PCR reactions are carried out in a reaction volume of 10 μl containing 1×GeneAmpe® 10×PCR buffer (Perkin Elmer P/N N808-0240), 13 ng of template DNA, 400 μM of each dNTP (Amersham Life Science Nucleix Plus™ 25 mM dNTP mix; Prod. No. US77119), 30 ng of each primer, 2 mM MgCl₂ and 0.5 u of AmpliTaq Gold™ polymerase (Perkin-Elmer P/N N808-0242).

[0081] Typical thermal cycling conditions using a Biometra UNO II cycler (Part No. 050-603; Anachem Ltd., Luton, UK) are as follows, the sequence Step 2-Step 3-Step 4 being repeated 36 times: Step 1 95° C. 10 min Step 2 92° C.  1 min Step 3 60° C.  1 min Step 4 72° C.  2 min Step 5 72° C. 10 min

[0082] To generate heteroduplexes, 2 μl of PCR product is denatured at 95° C. for 10 minutes and annealed at 68° C. for 30 minutes using a thermal cycler (eg. Biometra UNOII). 2 μl of 2×loading buffer (20% ethylene glycol, 30% formamide, 0.025% xylene cyanol, 0.025% bromphenol blue) is added to each sample before gel analysis.

[0083] A standard DNA sequencing apparatus (Owl Scientific S3S; Autogen Bioclear UK Ltd.) is used with a 60 sample comb (Owl Scientific S2S-60A; Autogen Bioclear UK Ltd.) and standard power supply (Biorad, Cat No. 165-5057) equipped with a temperature probe (Biorad, Cat No. 165-5058). A 0.4 mm thick 15% polyacrylamide gel is prepared using a 99:1 ratio of acrylamide to BAP cross linker, 10% ethylene glycol and 15% formamide in 0.5×TTE. Gels are pre-run for one hour at 30 watts, limiting the temperature to a maximum of 25ûC (using an electric fan to keep the temperature down if necessary eg. Jencons, Cat No. 292-004). After the pre-run, the wells are flushed with a pipette and the samples are loaded into the wells. The gel is then electrophoresed at 12 watts overnight (15 hours) at 25° C. Fragments greater than 350 bp remain on the gel.

[0084] After electrophoresis, the gel plates are separated. The gel is stained by placing the gel in 0.5×TTE containing lug/ml ethidium bromide (Biorad, Cat No. 161-0433) for 10 minutes, followed by destaining in 0.5×TTE for 10 minutes. The gel is then photographed on a UV transilluminator (eg. UVP GDS 7500).

[0085] Potential polynucleotide changes are detected by CSGE in five or more of the 16 patients for 9 of the 33 PCR fragments. For each of these potential changes, the PCR fragment from all 16 patients is subjected to double stranded DNA sequencing on an ABI377 automated sequencer using standard methods (http://www.pebio.com/ab/about/dna/377/) and the resulting DNA sequence is analysed using CONSED software (Gordon et al., Genome Res. 8:195-202) to confirm the presence of a sequence change and to identify the exact base change. All of the 9 potential changes detected by CSGE are confirmed. These changes are shown in the table below, in which #patients indicates the number of patients exhibiting the polymorphism: Alias Polymorphism # patients Prom 1.3 T to C 8 Prom 1.4 A to C 14 Intron 1.1 T to C 14 Intron 1.1 G to A 8 Intron 1.3 G to T 13 Intron 1.4 A to G 13 Intron 1.4 G to C 13 Exon 8.h C to A 7 3′.1 C to T 5

[0086] All nine polymorphisms are single nucleotide polymorphisms (SNPs). Several of the SNPs appear to be in linkage disequilibrium with each other as they occur with identical frequencies. In addition, the high frequencies also indicate a genetic association with the asthma phenotype in the patients and indicate that AAGB is an asthma susceptibility gene.

EXAMPLE 3

[0087] This Example relates to the expression of full length AAGB with a 6 histidine tag at the C-terminus using the Baculovirus system in T.ni Hi5 cells, and to the purification of the resulting polypeptide.

[0088] 1. Construction of a Recombinant AAGB Baculovirus

[0089] A unique EcoRI site is incorporated 5′ to the AAGB start codon by PCR amplification using the following primer: 5ST 5′-GAAGATCTTCG GAATTC CATC ATG GCGTTGGCGTTGGCGGCGCTG -3′

[0090] Another primer is used to introduce 6 histidine residues immediately prior to the AAGB stop codon. This primer also incorporates a unique KpnI site 3′ to the AAGB stop codon. 3ST 5′-AAGATCTTC GGTACC TTAATGGTGATGGTGATGGTGATAAATAAAGA GAACTCTG-3′

[0091] The recombinant “His tagged” version of AAGB is ligated as a 694 bp EcoRI/KpnI fragment into EcoRI/KpnI digested pFastbacl baculovirus transfer vector (Life Sciences). The recombinant AAGB sequence is transposed into Bacmid DNA carried by DH10Bac cells (Life Sciences; Bac to Bac Baculovirus expression system). AAGB recombinant Bacmids are isolated from DHlOBac cells and transfected into Sf9 cells using published protocols (Bac to Bac baculovirus expression system manual; Life Sciences).

[0092] 2. Amplification of Recombinant Baculovirus Stocks

[0093] The recombinant baculovirus is amplified by infecting Sf9 cells (maintained in SF900 SFMII medium; Life Sciences) at a cell density of 0.5×10⁶ cells/ml and a multiplicity of infection (mol) of 0.01 for 96 hours. Sf9 cells are then centrifuged at 1000×g for 5 minutes. The supernatants containing high titre virus are stored at 4° C.

[0094] 3. Expression of Recombinant AAGB in Hi5 Cells

[0095] Hi5cells (Invitrogen), maintained at densities of between 3×10⁵ and 3×10⁶ cells/ml in Excell 401 medium (JRH Biosciences; distributed by AMS Biotechnology in either shaker flasks (rotated at 90 RPM) or spinner flasks (stirring at 75 RPM) are infected with the amplified recombinant Baculovirus at a cell density of 2.0×10⁶ at an moi of 2.0 for 60 hours. Following infection Hi5 cells are centrifuged at 1000×g for 5 minutes, the supernatants poured off and the cell pellets frozen at −80° C.

[0096] 4. Crude Lysate Preparation

[0097] The cells (1×10⁹) are resuspended in 100 ml lysis buffer (20 mM Hepes pH 7.5, 100 mM NaCl, 5% glycerol, 2 mM beta-mercaptoethanol, 0.5 mM imidazole, 0.1% Nonidet P-40, 40 μg/ml AEBSF, 0.5 μg/ml leupeptin, 1 μg/ml aprotinin and 0.7 μg/ml pepstatin A). Cells are incubated on ice for 15 min then centrifuged at 39,000×g for 30 min at 4° C. The sample is filtered through a 0.22 μm filter immediately prior to use.

[0098] 5. Metal Chelate Affinity Chromatography

[0099] Metal chelate affinity chromatography is carried out at room temperature with a column attached to a BioCAD chromatography workstation. A 20 ml Poros MC/M (16 mmD×100 mmL) column is charged with Ni²⁺ prior to use and after each injection. To charge with Ni²⁺, the column is washed with 10 column volumes (CV) 50 mM EDTA pH 8, 1 M NaCl followed by 10CV water. The column is charged with 500 ml 0.1 M NiSO4 pH 4.5-5, washed with 10CV water, then any unbound Ni²⁺ removed by washing with 5CV 0.3 M NaCl. All steps are performed with a flow rate of 20 ml/min. The charged MC/M column is equilibrated with 5CV Buffer B (20 mM Hepes pH 7.5, 500 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, 1 mM PMSF, 5 mM imidazole) to saturate the sites followed by 10CV Buffer A (as Buffer B except 0.5 mM imidazole). 90-95 ml of the crude lysate is loaded onto the column per run at a flow rate of 20 ml/min. Subsequent steps are carried out with a flow rate of 30 ml/min. Any unbound material is removed by washing with 12 CV buffer A and AAGB eluted by applying a 0-50% Buffer B gradient over 10 CV. Fractions (8 ml) are collected over the gradient. AAGB-containing fractions are combined and protease inhibitors added to the final concentrations described for the lysis buffer above. DTT is also added to a final concentration of 1 mM. The combined fractions are dialysed overnight against 4 litres 20 mM Tris-HCl pH 7.5, 1 mM DTT, 0.2 mM PMSF at 4° C.

[0100] 6. Ion Echange (Anion Exchange) Chromatography

[0101] Resource™ Q chromatography is carried out at 4° C. with a column attached to an FPLC workstation (Amersham Pharmacia Biotech). A 6 ml Resource™ Q column (16 mmD×30 mmL) is equilibrated with 10 CV Buffer C (20 mM Tris-HCl pH 7.5, 1 mM DTT) at a flow rate of 2 ml/min. The dialysed metal chelate eluate is applied to the column and washed with 10 CV Buffer C. The protein is eluted by applying a 0-100% Buffer D gradient (20 mM Tris-HCl pH 7.5, 1 mM DTT, 1 M NaCl) over 10 CV. Fractions (3 ml) are collected on eluting the column.

[0102] 7. Gel Filtration

[0103] Del Filtration chromatography is carried out at 4° C. with a column attached to a BioCAD SPRINT chromatography workstation (PE Biosystems). A 318 ml (26 mmD×600 mmL) Sephacryl-200 prep (Amersham Pharmacia Biotech) column is equilibrated with 10 CV Buffer E (20 mM Tris-HCl pH7.5, 1 mM DTT, 150 mM NaCl) at a flow rate of 0.5 ml/min. The ion excahnge eluate is applied to the column and eluted with Buffer E. Fractions (1 ml) throughout the purification run are collected and analysed.

[0104] 8. Sample Concentration

[0105] Samples are concentrated approximately 10-fold using a Millipore Ultrafree-15 centrifugation device (MW cut-off 50 kDa) at 4° C. The device is pre-rinsed with water prior to use. The final storage buffer used for long term storage at −80° C. is 20 mM Hepes pH 7.5, 1 mM DTT, 100 mM NaCl, 5% glycerol. Glycerol can be omitted from the sample for storage at 4° C.

EXAMPLE 4

[0106] This example relates to the production of polyclonal antibodies against AAGB protein purified as described in Example 3. Polyclonal antibodies against a recombinant fragment spanning the extracellular domain are generated as described by Telo et al. (J. Biol. Chem. 273:17565-17572).

[0107] Immunisation of Rabbits

[0108] Dutch rabbits (Harlen-Olac) are immunised at 4 subcutaneous sites with 500 μg purified AAGB protein in PBS according to the following schedule: DAYS IMMUNISATIONS  0 1^(st) immunisation 1:1 in complete Freund's adjuvant 15 1^(st) boost 1:1 in incomplete Freund's adjuvant 45 2^(nd) boost 1:1 in incomplete Freund's adjuvant 55 1^(st) test bleed from the ear artery Every month Boost 1:1 in incomplete Freund's adjuvant until a good antibody response is obtained

[0109] Test bleeds (500 μl) are taken and the serum assessed for antibody titre. Serum is collected when a maximum titre is reached. This is done by collecting blood (10 ml) and allowing it to clot for 2 hours at 4° C. The blood is centrifuged at 1000×g for 5 minutes to separate the serum. The serum is removed and stored at −20° C. until assayed.

[0110] ELISA Screening

[0111] Nunc-Immuno Plate Maxisorp 96 well plates (Nunc, Fisher Scientific UK, Loughborough, UK) are used as a solid support and coated with the purified AAGB protein (100 ng/well) overnight at 4° C. The plates are blocked for 3 hours at 37° C. with PBS containing 2% BSA (Sigma) and 0.02% NaN₃ (Sigma). After blocking, plates are incubated overnight at room temperature with serum in different dilutions of PBS. The presence of polyclonal5 antibodies is checked with both biotin labelled IgG-antibodies to rabbit (Goat anti-rabbit IgG antiserum, 1:25000 dilution), with an incubation time of 40 min. Alkaline phosphatase conjugated streptavidin (Immununo Research, Dianova, CH) is then added at a dilution of 1:10000. Development of the reaction is carried out by adding an alkaline phosphatase substrate (Sigma, f.c. 1 mg/ml) dissolved in diethanolamine. After 45 min. absorbance is read at 405 nm with a reference of 490 nm with an ELISA plate reader (Bio-rad laboratories Ltd., Hemel Hempstead, UK).

[0112] Purification

[0113] 5 ml protein A-agarose is poured into a chromatography column and washed with 6 column volumes of 0.1 M tris (hydroxymethyl) methylamine (Tris) buffer pH 7.5. The rabbit serum containing anti-AAGB antibodies is diluted (½) with Tris buffer and added to the protein A-agarose. Unbound proteins are removed by washing the column with 6 volumes of Tris buffer. The IgG is eluted off the column with three column volumes of 0.1 M glycine buffer pH 3.0 and collected as 1 ml fractions into tubes containing 28 μl of 1 M Tris. The fractions which are positive for protein content are checked for purity by SDS-PAGE under reducing conditions. Two bands at 50 and 25 Kd are visualised corresponding to the heavy and light chains of an immunoglobulin molecule. Fractions containing only immunoglobulin are pooled, re-checked for protein concentration and stored at −20° C.

EXAMPLE 5

[0114] This exaple describes the preparation of monoclonal antibodies against AAGB protein purified as described in Example 3.

[0115] Immunisation od Mice

[0116] Female Balb/c mice are immunised intraperitoneally with 100 μg of AAGB protein in PBS according to the given below: DAYS IMMUNISATIONS  1 1^(st) immunisation 1:1 with complete Freund's adjuvant 14 1^(st) boost 1:1 with incomplete Freund's adjuvant 21 2^(nd) boost 1:1 with incomplete Freund's adjuvant 28-30 Three final boosts in PBS 31 Fusion with mouse myeloma cells

[0117] Serum is assessed for antibody titre by ELISA (Example 4) after the animal is sacrificed for the preparation of spleen cells for fusion. If antibody titre is sufficient, ({fraction (1/1000)} to {fraction (1/100,000)}), the hybridomas are screened, otherwise discarded.

[0118] Preparation of Myeloma Cells

[0119] Sp2/0 murine myeloma cells (ATCC #CRL 1581; maintained in culture medium containing 20 μg/ml 8-azaguanine) are cultivated for one week before fusion in RPMI 1640 (8-azaguanine is not included), 10% (v/v) FCS and 1% penicillin-streptomycin (50IU/ml and 50 μg/ml, respectively). The cells are harvested by centrifugation (200×g for 5 min) and washed three times in cold RPMI 1640. Approximately 2.5×10⁶ cells are used per 96 well microtitre plate.

[0120] Preparation of Spleen Cell Suspension

[0121] The mouse is killed by an overdose of anesthetic (Forene), the spleen dissected and pressed through a cell strainer (70 μm mesh cell strainer; Becton & Dickinson, Oxford, UK, Cat. No 2350). The cell suspension is washed three times in RPMI 1640 (as above) and counted: 5.10⁶ cells /96 well plate are necessary.

[0122] Fusion of Myeloma Cells and Spleen Cells

[0123] The spleen and myeloma cells are mixed (2:1), centrifuged (200 ×g for 5 min) and the pellet warmed in a 37° C. water bath. Prewarmed polyethylene glycol 4000 (1 ml per 10⁸ cells) is added slowly over one minute, then 20 ml of prewarmed wash medium over two minutes. After centrifugation the pellet is carefully resuspended in selection medium (RPMI 1640, 10% FCS, 1% penicillin-streptomycin, 10% BM condimed H1 (feeder cell replacement from Boehringer Mannheim, Lewes, UK; Cat. No. 1 088 947), 10% HAT-media supplement (hypoxanthine, aminopterin and thymidine to select against unfused myeloma cells; Boehringer Mannheim, Lewes, UK; Cat. No. 644 579) and plated, 200 μl/well of a 96 well microtitre plate.

[0124] After five days clusters of hybrid cells can be identified by examining the bottom of the microtitre wells with an inverted microscope. After 10-14 days the culture supernatant is tested for the presence of antibodies by ELISA (example 4). The positive clones are expanded in a 24 well assay plate and retested.

[0125] Cloning of Positive Hybridomas

[0126] The expanded clones which are still positive are cloned by limiting dilution. Cells are diluted serially in four dilutions steps in a 96 well microtitre plate; 5, 2, 1 and 0.5 cells/well. HAT-media supplement is replaced with HT-media supplement (Boehringer Mannheim, Lewes, UK; Cat. No. 623 091). After approximately one week the cells are screened by ELISA (Example 4). The cells of those wells containing a single positive clone are expanded.

[0127] Production of Monoclonal Antibody Supernatant

[0128] The cells are grown in culture flasks in standard medium (RPMI 1640, 10% (v/v) FCS and 1% penicillin-streptomycin) until the hybridomas overgrow and die. The debris is removed by centrifugation and the supernatant containing the antibodies is titred using ELISA (Example 4) before storing under sterile conditions at 4° C., −20° C. or −70° C.

1 84 1 2057 DNA Homo sapiens misc_feature (1)...(2057) n = A,T,C or G 1 ggatcctcgg cggcggcggc ggtgcttaca gcctgagaag agcgtctcgc ccnggagcgg 60 cggcggccat cgagacccac ccaaggcgcg tccccctcgg cctcccagcg ctcccaagcy 120 gcgrccgcgc cccttcagct agctcgctcg ctcgctctgc ttccctgctg ccggctgcgc 180 atggcgttgg cgttggcggc gctggcggcg gtcgagccgg cctgcggcag ccggtaccag 240 cagttgcaga atgaagaaga gtctggagaa cctgaacagg ctgcaggtga tgctcctcca 300 ccttacagca gcatttctgc agagagcgca ssatattttg actacaagga tgagtctggg 360 tttccaaagc ccccatctta caatgtagct acaacactgc ccagttatga tgaagcggag 420 aggaccaagg ctgaagctac tatccctttg gttcctggga gagatgagga ttttgtgggt 480 cgggatgatt ttgatgatgc tgaccagctg aggataggaa atgatgggat tttcatgtta 540 acttttttca tggcattcct ctttaactgg attgggtttt tcctgtcttt ttgcctgacc 600 acttcagctg caggaaggta tggggccatt tcaggatttg gtctctctct aattaaatgg 660 atcctgattg tcaggttttc cacctatttc cctggatatt ttgatggtca gtactggctc 720 tggtgggtgt tccttgtttt aggctttctc ctgtttctca gaggatttat caattatgca 780 aaagttcgga agatgccaga aactttctca aatctcccca ggaccagagt tctctttatt 840 tattaaagat gttttctggc aaaggccttc ctgcatttat gaattctctc tcaagaagca 900 agagaacacc tgcaggaagt gaatcaagat gcagaacaca gaggaataat cacctgcttt 960 aaaaaaataa agtactgttg aaaagatcat ttctctctat ttgttcctag gtgtaaaatt 1020 ttaatagtta atgcagaatt ctgtaatcat tgaatcatta gtggttaatg tttgaaaaag 1080 ctcttgcaat caagtctgtg atgtattaat aatgccttat atattgtttg tagtcatttt 1140 aagtagcatg agccatgtcc ctgtagtcgg tagggggcag tcttgcttta ttcatcctcc 1200 atctcaaaat gaacttggaa ttaaatattg taagatatgt ataatgctgg ccattttaaa 1260 ggggttttct caaaagttaa acttttgtta tgactgtgtt tttgcacata atccatattt 1320 gctgttcaag ttaatctaga aatttattca attctgtatg aacacctgga agcaaaatca 1380 tagtgcaaaa atacatttaa ggtgtggtca aaaataagtc tttaattggt aaataataag 1440 cattaatttt ttatagcctg tattcacaat tctgcggtac cttattgtac ctaagggatt 1500 ctaaaggtgt tgtcactgta taaaacagaa agcactagga tacaaatgaa gcttaattac 1560 taaaatgtaa ttcttgacac tctttctata attagcgttc ttcaccccca cccccacccc 1620 cacccccctt attttccttt tgtctcctgg tgattaggcc aaagtctggg agtaaggaga 1680 ggattaggta cttaggagca aagaaagaag tagcttggaa cttttgagat gatccctaac 1740 atactgtact acttgctttt acaatgtgtt agcagaaacc agtgggttat aatgtagaat 1800 gatgtgcttt ctgcccaagt ggtaattcat cttggtttgc tatgttaaaa ctgtaaatac 1860 aacagaacat taataaatat ctcttgtgta gcacctttta aaaaaaaaaa aaaaaaaaaa 1920 mwvgcctgcc attttggttg attgccnagg caaatttttt ccnaatccta ngggaatcnt 1980 tccaggtaga tgcgattaaa aaanccaatg gtnggggcca atttttttcc tccattttca 2040 ggcnccaaga aggcccc 2057 2 725 DNA Homo sapiens 2 tttttttttt tcatttttta acaagcaaat tttaataatg ccttttattt ctatacaaag 60 caatgtaact ttctgaaaaa aaaaatggct atacagaacc ctttaaacat aagagtacag 120 agtttcaaat ggcaacaaga agttaagaaa catagggcac tgtgtcgtta tgggtgaatc 180 ctagtcgtcc tgcagcccaa ggtccaagct agtttactcc ataaccttaa gtaaataacc 240 gcggttccta tgaatacctt tccaaaacca tttattataa aaactcactc tttatccatt 300 atcagtatta acgtatcaaa gtgagaaaga acatgggaga gaaaaatgac aattaatgca 360 gcgtgaacat tttcatcctg aataccagta aggactctag atgatatatg gtgcccactt 420 catgtgcctc aaaagatttt aattttcaga acatttaaac gattaaagag aaatcatcct 480 cttaactagg tattttaaag cktatcaaac tgtaggttta catttgacaa agtcttatgg 540 accaccaaat acgttrgaaw taggaattcc aaatatttca gttcgttgaa tatacaaaac 600 caaataaggc gcaaggacct gaagaataaa cacaaagcta gtctacaatg tttgagttaa 660 cgtgatttag gacccttcta ttttgttctg caagataatt tacatagttc tctctgctat 720 atgtg 725 3 3599 DNA Homo sapiens 3 gcggcggctg cggtgcttac agcctgagaa gagcgtctcg cccgggagcg gcggcggcgg 60 tgcttacagc ctgagaagag cgtctcgccc gggagcggcg gcggccatcg agacccaccc 120 aaggcgcgtc cccctcggcc tcccagcgct cccaagccgc agcggccgcg ccccttcagc 180 tagctcgctc gctcgctctg cttccctgct gccggctgcg ccatggcgtt ggcgttggcg 240 gcgctggcgg cggtcgagcc ggcctgcggc agccggtacc agcagttgca gaatgaagaa 300 gagtctggag aacctgaaca ggctgcaggt gatgctcctc caccttacag cagcatttct 360 gcagagagcg cagcatattt tgactacaag gatgagtctg ggtttccaaa gcccccatct 420 tacaatgtag ctacaacact gcccagttat gatgaagcgg agaggaccaa ggctgaagct 480 actatccctt tggttcctgg gagagatgag gattttgtgg gtcgggatga ttttgatgat 540 gctgaccagc tgaggatagg aaatgatggg attttcatgt taactttttt catggcattc 600 ctctttaact ggattgggtt tttcctgtct ttttgcctga ccacttcagc tgcaggaagg 660 tatggggcca tttcaggatt tggtctctct ctaattaaat ggatcctgat tgtcaggttt 720 tccacctatt tccctggata ttttgatggt cagtactggc tctggtgggt gttccttgtt 780 ttaggctttc tcctgtttct cagaggattt atcaattatg caaaagttcg gaagatgcca 840 gaaactttct caaatctccc caggaccaga gttctcttta tttattaaag atgttttctg 900 gcaaaggcct tcctgcattt atgaattctc tctcaagaag caagagaaca cctgcaggaa 960 gtgaatcaag atgcagaaca cagaggaata atcacctgct ttaaaaaaat aaagtactgt 1020 tgaaaagatc atttctctct atttgttcct aggtgtaaaa ttttaatagt taatgcagaa 1080 ttctgtaatc attgaatcat tagtggttaa tgtttgaaaa agctcttgca atcaagtctg 1140 tgatgtatta ataatgcctt atatattgtt tgtagtcatt ttaagtagca tgagccatgt 1200 ccctgtagtc ggtagggggc agtcttgctt tattcatcct ccatctcaaa atgaacttgg 1260 aattaaatat tgtaagatat gtataatgct ggccatttta aaggggtttt ctcaaaagtt 1320 aaacttttgt tatgactgtg tttttgcaca taatccatat ttgctgttca agttaatcta 1380 gaaatttatt caattctgta tgaacacctg gaagcaaaat catagtgcaa aaatacattt 1440 aaggtgtggt caaaaataag tctttaattg gtaaataata agcattaatt ttttatagcc 1500 tgtattcaca attctgcggt accttattgt acctaaggga ttctaaaggt gttgtcactg 1560 tataaaacag aaagcactag gatacaaatg aagcttaatt actaaaatgt aattcttgac 1620 actctttcta taattagcgt tcttcacccc cacccccacc cccacccccc ttattttcct 1680 tttgtctcct ggtgattagg ccaaagtctg ggagtaagga gaggattagg tacttaggag 1740 caaagaaaga agtagcttgg aacttttgag atgatcccta acatactgta ctacttgctt 1800 ttacaatgtg ttagcagaaa ccagtgggtt ataatgtaga atgatgtgct ttctgcccaa 1860 gtggtaattc atcttggttt gctatgttaa aactgtaaat acaacagaac attaataaat 1920 atctcttgtg tagcaccttt tactgtagat tagtgcttaa tttcttggct tgcatttgtt 1980 gattgctaag gcaatttttt ctaatcttag ggaatcattc agtagatgcg attaaaaaac 2040 taatgttggg tcaatttttt tcttcatttt cagcacaaga agtcctctta tatcctacta 2100 aatacattcc taaaaatgta tttgaacatt ggttctgtaa aagataatgg actaaaaaag 2160 tagagaggag ttgtagagat cttaaatcat tctggaattc ctaattatgc ttcaattttt 2220 agacataatt ttagataatt tatttccagt gttttctgca tgttctcatt tgttcttttt 2280 ctcagttgaa tgcaccaact ggtttgagtc ctgtgagcat tcagtcagtt gaaattaaag 2340 attcctcatt tctcctgatt tctattcttg tctcaatctt aaatttagag accagttgtt 2400 tttatgatat cagccatttg atttttttca ttttctattt aagaaatatg aagaaaaaat 2460 acaccaagat ggtcaaatta ctacacaaat cagcaccagc acagtctgat agctgcaaat 2520 gtccattcat ctgctgtgta tgtatatcca gaatcagcat aggaagtcgt tcaggatatc 2580 agtatataat gcacagaagt gtgggttgtt tgaaagccaa acaggaaaat taggagcctc 2640 ctggattgac atttcagtga tccctctaac cagtttatgg attattatga ataatagtgt 2700 agtgtgttct ttttcagaag ttatatttga taatagagaa gggagtttta tggaagtttc 2760 tttgaagatt tttttttttc catttcgaat cagattatag caacaatgga gtttggaagt 2820 ttgtatggcc tataatgttc taagttccag aatgaaaaga tctgtaacaa tctgaataga 2880 tgtggacaca tatagcagag agaactatgt aaattatctt gcagaacaaa atagaagggt 2940 cctaaatcac gttaactcaa acattgtaga ctagctttgt gtttattctt caggtccttg 3000 cgccttattt ggttttgtat attcaacgaa ctgaaatatt tggaattcct atttctacgt 3060 atttggtggt ccataagact ttgtcaaatg taaacctaca gtttgataag ctttaaaata 3120 cctagttaag aggatgattt ctctttaatc gtttaaatgt tctgaaaatt aaaatctttt 3180 gaggcacatg aagtgggcac catatatcat ctagagtcct tactggtatt caggatgaaa 3240 atgttcacgc tgcattaatt gtcatttttc tctcccatgt tctttctcac tttgatacgt 3300 taatactgat aatggataaa gagtgagttt ttataataaa tggttttgga aaggtattca 3360 taggaaccgc ggttatttac ttaaggttat ggagtaaact agcttggacc ttgggctgca 3420 ggacgactag gattcaccca taacgacaca gtgccctatg tttcttaact tcttgttgcc 3480 atttgaaact ctgtactctt atgtttaaag ggttctgtat agccattttt tttttcagaa 3540 agttacattg ctttgtatag aaataaaagg cattattaaa atttgcttgt taaaaaatg 3599 4 221 PRT Homo sapiens 4 Met Ala Leu Ala Leu Ala Ala Leu Ala Ala Val Glu Pro Ala Cys Gly 1 5 10 15 Ser Arg Tyr Gln Gln Leu Gln Asn Glu Glu Glu Ser Gly Glu Pro Glu 20 25 30 Gln Ala Ala Gly Asp Ala Pro Pro Pro Tyr Ser Ser Ile Ser Ala Glu 35 40 45 Ser Ala Ala Tyr Phe Asp Tyr Lys Asp Glu Ser Gly Phe Pro Lys Pro 50 55 60 Pro Ser Tyr Asn Val Ala Thr Thr Leu Pro Ser Tyr Asp Glu Ala Glu 65 70 75 80 Arg Thr Lys Ala Glu Ala Thr Ile Pro Leu Val Pro Gly Arg Asp Glu 85 90 95 Asp Phe Val Gly Arg Asp Asp Phe Asp Asp Ala Asp Gln Leu Arg Ile 100 105 110 Gly Asn Asp Gly Ile Phe Met Leu Thr Phe Phe Met Ala Phe Leu Phe 115 120 125 Asn Trp Ile Gly Phe Phe Leu Ser Phe Cys Leu Thr Thr Ser Ala Ala 130 135 140 Gly Arg Tyr Gly Ala Ile Ser Gly Phe Gly Leu Ser Leu Ile Lys Trp 145 150 155 160 Ile Leu Ile Val Arg Phe Ser Thr Tyr Phe Pro Gly Tyr Phe Asp Gly 165 170 175 Gln Tyr Trp Leu Trp Trp Val Phe Leu Val Leu Gly Phe Leu Leu Phe 180 185 190 Leu Arg Gly Phe Ile Asn Tyr Ala Lys Val Arg Lys Met Pro Glu Thr 195 200 205 Phe Ser Asn Leu Pro Arg Thr Arg Val Leu Phe Ile Tyr 210 215 220 5 1054 DNA Homo sapiens 5 gcaaaggcct tcctgcattt atgaattctc tctcaagaag caagagaaca cctgcaggaa 60 gtgaatcaag atgcagaaca cagaggaata atcacctgct ttaaaaaaat aaagtactgt 120 tgaaaagatc atttctctct atttgttcct aggtgtaaaa ttttaatagt taatgcagaa 180 ttctgtaatc attgaatcat tagtggttaa tgtttgaaaa agctcttgca atcaagtctg 240 tgatgtatta ataatgcctt atatattgtt tgtagtcatt ttaagtagca tgagccatgt 300 ccctgtagtc ggtagggggc agtcttgctt tattcatcct ccatctcaaa atgaacttgg 360 aattaaatat tgtaagatat gtataatgct ggccatttta aaggggtttt ctcaaaagtt 420 aaacttttgt tatgactgtg tttttgcaca taatccatat ttgctgttca agttaatcta 480 gaaatttatt caattctgta tgaacacctg gaagcaaaat catagtgcaa aaatacattt 540 aaggtgtggt caaaaataag tctttaattg gtaaataata agcattaatt ttttatagcc 600 tgtattcaca attctgcggt accttattgt acctaaggga ttctaaaggt gttgtcactg 660 tataaaacag aaagcactag gatacaaatg aagcttaatt actaaaatgt aattcttgac 720 actctttcta taattagcgt tcttcacccc cacccccacc cccacccccc ttattttcct 780 tttgtctcct ggtgattagg ccaaagtctg ggagtaagga gaggattagg tacttaggag 840 caaagaaaga agtagcttgg aacttttgag atgatcccta acatactgta ctacttgctt 900 ttacaatgtg ttagcagaaa ccagtgggtt ataatgtaga atgatgtgct ttctgcccaa 960 gtggtaattc atcttggttt gctatgttaa aactgtaaat acaacagaac attaataaat 1020 atctcttgtg tagcaaaaaa aaaaaaaaaa aaaa 1054 6 50000 DNA Homo sapiens 6 cttcacccct tagtagctct gtgtcattgg gtgagatatt taatttcttt gggcataact 60 tcctcatctg tgaaacactg ataattgtat ctcctatatg gcagctatgc cagcctcctt 120 tctgttcttc cacaagacaa tctctctctg gaccgccctt tgcacctgtt cctgtggtcg 180 ggaaagcctt tagcatggcc aaacccttca agttttgact tcatgtcacc gagtcaggga 240 gcttttttgg aaaatattgt ttccgattcc gcagatgagg ctgttaaaat actaaaggat 300 ggcataaaca tgaggatgct attataatca tccaccctaa gtaggatttg agatccaagc 360 agttctcaag gatttagggt tagcaaactt tttctgtaaa atgtcagata gcaagtatta 420 tagggtttgc aggccatacc aaacaaatgg gtgctgctgt gttccaataa aactttattt 480 atggacactg aaacttgaat ttcatgtact tttcacagtc atggtggcag accggatttg 540 gccccctccc tgtcgctggc tgacccctgg tttatgagta cggcagataa aagaagggag 600 tgaataagaa ccacgattct gctgccagac acgagggatc aggtcgctgc tgtgaagatt 660 gtcaacctct ctggttgcca acgacctcga gaatggggtt gtggtccctg cgtcagggcg 720 gttgtgctga aggctggatg agggcacgta ggtgaagctc agtgcttggc acgaggtgct 780 catcagcagc atcgcccttg ctttgggctg gcctgttagt aattcgtttt tagtggaatc 840 gaatacaaca cttgagcctc ggaggctcag gtcgtcggag caactacacc agggccggtg 900 cacctcgggg cggcggcgca ggactaggct aggccgggcc gggccgggca gggcggcgcg 960 agtcgggggt ggggcggcgg cgcagcgccg tcgtcgtagt ccccgcctct tccccagggg 1020 ccgcgtcgga gcctcggcgg cggcggcggt gcttacagcc tgagaagagc gtctcgcccg 1080 ggagcggcgg cggccatcga gacccaccca aggcgccgtc cccctggact tccagcgcta 1140 ccaagcagca acgtccgcgc cccttcagct agctcgctcg ctcgctctga ttccctgctg 1200 tcgactgcgc catggcgttg gcgttggcgg cgctggcggc ggtcgagccg gcctgcggca 1260 gccggtacca gcaggtaagc ggcgcccgac tccagccccg aactccggtc cctggctctg 1320 ccctgcccgc tggccgcctc aggcctctct ggccggcccg cggccaactc gacgccgggg 1380 cttggcaggc cgcgctgggc ctggaggggc gggtcgggcc ccgggctcgg attcgaggct 1440 gccacgggcc cggcctgccc gtctgcgacc ccaggtggga ctccggcggt ggaaagtcct 1500 cctcccggcc tcgtttcccg catttcccag ggcgggcggg tccctgagtc agaacccgcg 1560 acccacaggc ggcatcgatc tgaggcattc gtttttcatt tttccacctt tggtttgatg 1620 tcattggaaa agaaagaaag aaaaaagcat gaaacggaaa ctgggtggag agaaaagatt 1680 aaaatctctg ctgctgccga tagctcccca cagcaagatg ctattgctca gggtttcaga 1740 gcctagtttg tcgaaaccca tccccctcgc ctccgtttta agacaaaagt aaaaaatgaa 1800 gcaagagaca agcccgcagt ttgcagtaga tttggaagag gcgatgtttg gctgtggtga 1860 tgccccaaaa gggagagcta cgatgaagtc tgcagggcgt gtgccgggtg tattttaaat 1920 gcttggaagt gacgttttaa gagatttgat ggcatagctg gggaggaaat gtataggaaa 1980 tgattggatc attttctcgg gtttgattta atacattgag gccccaggga aaagagctgc 2040 ttgtttcact ccgcgtctgt agctaagttc ccccaggaaa tttagagacc ttctgcaggg 2100 tttttggaag tgttaatcta acatacagta atggtgggaa ttgagccagg tggtaaactg 2160 atgttgattg gaaccgcaaa taacggcttg tgtttgtcat gtttcagatg ctttcggtgc 2220 atacagatgt caatcctcct tttatcaaat aaaatgtcct atcttagacg atctacaatt 2280 ttatatttta gaagtaagga aagccagcca gtacgatgaa aacgtagatt aaccctggtg 2340 cggccggccg gttactggtt tgttttgcta tcatcagcca tgtgttgcaa tccgtcccca 2400 ctcaggcttg gactcccaaa atgactagtt aagaatttca cttcttggtt ttataaacac 2460 ggcccaaatt gtggcaatgc agttaatcct tagatctctt cagagcacat ggatctagca 2520 catgcttttt ttctatgacc ttgggcagcc tgcgggttcc ttactgcttt aataaaatgg 2580 gaataacaga tgccacttaa ctgtcaccac agtgtttgca aatcacccct actgtatact 2640 gtcatctgat ttcatccttt tcatcctgac ttggataagg ctaacgagca aaagagctgt 2700 ggtttttctt tgtactgcca tcttcttgag ctattaggta atgctgacat actgctagaa 2760 tataaaaaat ttgctggagt atcaaaatca tgtattcctt agtgtggcat ttaatatttt 2820 ttaattatgc agaattgaag tataaaaaaa agagaaaaaa atcttcctag aaaaattgta 2880 aaagatgaaa gcccaaaaaa ctgcaaaacc ctccctaaac aaaaaccaaa tagataacca 2940 aaggtaatat tttaatgtgc attcttgtgg gtttatagct atgtaaatat acatagttat 3000 agatcagtat atgtgtgtac agataaggaa tgcaggaaag ctaatattga tttttttatg 3060 caacaatcac agtattaagt aatttacatc catttttttt ttcttttaat tcataccaaa 3120 cccttatgaa ccagggtttg ttctcattgt caaagactca aagtggctca gtaacttgcc 3180 tcaaggtcac aggctgctaa gtagtgatta taaggatata cacacactca cctacttaaa 3240 acacaggctc acagtccaga tataggtgta tgcattagca gatatataca catgcatatg 3300 tattctctta tgtacccata tatacgtata cagacatatc tttgtacatt tgtgtgcagt 3360 tttatgagcc ccgatcatac ttgcacatac ttttgcacgt gtccatgtgt atcttgttct 3420 cagacatgtg cattcataca tttgcacatg tgtatcatgc acatgtatct acaaaggcat 3480 gctagataaa taatgaaata ggttcataat tctattttgt aatgttcttc acacaatgtt 3540 attaatgtct tctatataaa atatttttaa tgtctgcatg gcatgtaatt gtatggctga 3600 atcataattt aactaattgc ttatctttgg tcactaagaa tgtttacagt ttttttcatt 3660 gaaatttatt tcacatacgt cttcaaccgt ttccttagca taaattacta gccatggaaa 3720 tgcctggtca gcgaatatgc atttttaaaa ggcttttgaa ttatagaatg atggtgaatt 3780 gtccaggcaa cttgatagga gcatgatact ctttggttgt cacacagggc tgttctgact 3840 tctatttgat ctggttaatc aaattgagct agttttctca ccaagtaaat gtaatggaaa 3900 actgtatgat tcattcataa atgcaaacac agttttgtct ttatggtctt tttattggta 3960 gtgtaatatg tggacaactt gaatttagca tagtgacaga ctatgcagaa agggtaggct 4020 caaccaggtg tggtggctca cacctgtaat cacagcactt tgggaggccg aggtgggcgg 4080 atcacttgag gtcaggagtt cgagaccagc ctggccaaca tggcaaaacc ttgcctctac 4140 taaaaataca aaaattagcc aggtgtggtg gcactcacct gtaatcccag ctacttggga 4200 ggctgaggca ggagaattgc ttgaacctgg gaggcagaag ttgcagtgag ccaagattgt 4260 gccactgaac cccagcctgg gtgacagagt gggactgtct caaaaaaaac caaaacaaaa 4320 caaaacaaaa aatggccagg caaggtggct catgcctgta atcccagcac tttgggaggc 4380 caaggcaggc agatcacaaa gatcacgagg tcaagagatt gagaccatcc tggccaacat 4440 gtgaaacccc atctctatta aaaatacaaa aattagctgg gcgtggtggc gtgcacctgt 4500 agtcccagct actcaggagg ctgaggcagg agaatcgctt gaacctggga ggcagatgtt 4560 tcagtgaact gacatcgtgc cactgcactc ctgcctggcg acagagcaag actctgttta 4620 aaaaaaaaag cacggtgtgg tggtgcacat ctgtaactcc agctacttgg gaggctgagg 4680 caggaaaatc acttgaaccc aggaggcaga gtttgcagta ggctgggatc gcgccactgt 4740 actccaggtt gggtgagcaa gactgtctta aaaaaaaaaa aaaaaaaagg caccgaggtg 4800 ggcggatcac aaggtcagga gatcgagacc atcctggcta acatggtgaa accccatctc 4860 tactaaaaat acaaaaaatt agttgggcgt ggtggcacgt gcctgtagta gtcccagcta 4920 ctcgggaggc tgaggcagga gaaacccggg aggtgaagat tgcagtgagc cgagatcgcg 4980 ccactgcact ccaggctggg cgacgaagtg agactctgtc caaaaaaaaa aaaaagaggc 5040 taggcttata ggttcccctg gcattatttt attttttttt tcatttttgc cttttgaaac 5100 tgagactgtc tcagacagta tggaagcact attttagaaa gacctaagta ggcccatact 5160 gagtgctgag aaacctgttg cgtgtttctc tctctgagga cgttaactcc cgttcatcca 5220 tagatgaacg cagatcttct gcggtagatg tgtgcatgaa ttttgccaat tcatttcaag 5280 tattttggga tacttggatt tgaattcaat gtaagtgggt gatgatgaca ctgccatgta 5340 ggttcaaaca gattacagaa tgtttagttc tggacttgcc tcattacctg ctaaagaagt 5400 tgcccacttg tgagcatcac caatactctg tatgatagtt tgatgacatt atagtgcaga 5460 ttttggaatg gtatactttt ggataaattc tctcaggatt atggaggaat attttgatga 5520 gcataagaat ttcatctctg ataggtcaaa ccatattcat gttaaattat tagatgaagt 5580 aatgatcctg gaggaaggaa tgaatagtgc ttatgtggct tttttgctta tttttcagtt 5640 tcaaaagaag ttatatcaca gaatgccaac ataacctaaa tgtcaaagtt tcttaagtta 5700 tatgctgtat ttgtggccta catggtcagt gatcaaagaa aatttataaa aatattagga 5760 agatgggtaa attattcctt tgaaaacttt aattgtggta aaatacacat aaaatttacc 5820 atgttaacca cttttaagta tacagtttag tggcattaag tacattcaca ttgttgtgga 5880 accagtccat ctccagaact ttgtcacctt gcagaactga aactccatat ccattaaaca 5940 ataactctcc attccccctc ctccagcccc tggaagccac cattctactt tctatgtatt 6000 tgactaccgt aggtacctct cataagtgga attagacagg atttgtcctt ttgtgactgg 6060 catatgtcac ttagcataat gtcttcagag ttcatccatg ttgtagcatg tgtcagaatt 6120 tcctgcctct ttaaggctga ataatagtcc attttatata tgtaccacat tttgtttatg 6180 cattcatttg ttgacggatg cttggtttgc ttctaccttt tggtcattgt gagtaatgct 6240 gctataaaaa acatgagtct acaaatatct ttcaagtccc tgctttcagt tcttttggat 6300 atacccagaa atggaattgc tgggtcatac ggcaattcta tttttaagtt ttcgagaaac 6360 tcccatactg ttttccatgg cagcttcacc attttatttt cccaccaaca atgcataagg 6420 attccaattt ctccatatcc tcaacatact tgttatgttc tcttttttgt ttttgttttt 6480 ttgatagtag ccatctaatg ggtgtgaagt ggtatctcat tgcagtattg atccctattt 6540 ccctaataat tagcgatgtt gagcatcttt tcatgtgctt attggccatt gatattttct 6600 ttagagaaaa tgtgtattca tgttctttgt tcatttttga attgagtttt ttgttgtcga 6660 gttgtaggag taatttcttc atgatgttta cattttcaac tttttactat ggaaatttca 6720 aacacataca ataataaagt agtgtaagaa attttatgta ccatcaccca gcttcagcaa 6780 ttgtgaatga atggccaata ttatttcact tatactcacc acattatttt gaaacgaatc 6840 ctagaaatca tatattgata aatatttcta tgtgtaacgc taaaagaaaa caaatggtat 6900 gatatttcaa gaagatatca catgaggaat atatcatgag agctctcttg taatctgtga 6960 ggagtatgga aacaggattt gtcagtgtcg ggctgtggat ccataactaa ctgtcctttg 7020 tattgattgg gctcactctt gccaagcctc atgctttgtt tttgtagcag tgatagattc 7080 ctgttaatct atccttgttt tcttacctga tcccatgggt gaccacttgt taggcaagat 7140 ttatttggct taatcatact atctgtgtcc tgaaaatagg gcagtgcttc ctggtttggg 7200 cagaaaccaa ggcccagttg aactgctcta gggaattttg ctctaagctg atgccaccag 7260 tagtttaact gcacataact tcaaattgtt ttatcctaga cattaattat atttcataat 7320 tttatggtag attataaagt aaatcctacc atgagaataa cttcatattt ttattaagaa 7380 agaaaaaaag tctccaagag attcaagtga catcctcaga tttatttgtt tttagggctt 7440 taaaataatt attttggttt cttaaaatgt tcttgtttta cctttaagca agtacaacag 7500 cttagaagaa ttatattctt ttttgtttgt ttgttttgag atggagtgtc tttcttcttg 7560 ctcaggctgg aaggcaatgg cgcgatctcg gctcactgca acctccgcct cccaggttca 7620 agcgattctc ctgcctcagc ctcctgagta gctgggatta caggtgccca ccaccacgcc 7680 cagctaattt tttgtatttt tcgtagagat ggggtttcac catgttggcc aggctggtct 7740 tgaactcctg acctcaagtg atctgcccgc ctcggtctcc caaagtgctg ggattacagg 7800 tgtgagccac tgcgcccggc ctatattctt ataataatgg acaatttaga taaagcaact 7860 ttaatacttc tgttccattc atactgaaaa ttggcatgat ggtttatctt tttttcaagt 7920 cattgttgcc aataagctag gcaactggca aacatcatgt atgaaatttt ttgggcttga 7980 catactttcc agtggtaatt gcttctagtg gttggctagg tgggggtatg agttagagta 8040 ggctctgggt atccacaggt tctacattcg cagattcaac caactgcaga ttgaaaatag 8100 gggaaaataa ccatacaata ataaataata gtgcaagtaa aatatacact ataacagcta 8160 ttttacatag catttacatt gtattaagtg ttataagtaa agatgattta aagtacatga 8220 ggtaggttat gtgtaaatac tataccattt tatgtcagag gcttgtgcat cctcccattt 8280 tggtatctgt ggggagtcct ggaaccaatt cccagtggat actgagggat gactgtatga 8340 tatgtcttgt taacttgacc aagagtggta gtaaagcaag tgtaacttga tatcacttag 8400 cacatcctgc tgttacaata aggcttactc atgatttttc tgaataacat aatttatatt 8460 aaatgtccta aatactagca ctgatcaagt ttaagagcat tcctagtgaa atattttctt 8520 attaatgcag ttcccttcat atgtatattt atttatttat ttccttcctt ccttccttcc 8580 ttccttcttt ctctctctct ctctctctct ttctttcttt ctttcaacag agtcttgctc 8640 tgttccccag gctggagtgc agtggcgcaa tcttggcaca ctgcaacccc tgccttccag 8700 tttgaagcca ttctcctgcc tcagcctctg gagtagctgg gactacaggt gcccgccagc 8760 aggcccggct aatttttgta tttttagtag aggtggggtt tcaccatgtt ggccaggcta 8820 gtctcgaact cctgacttca aatgatctac ccaccttggc ttacagagat tacaggtgtg 8880 agccaccatg cccaggctac cttcatatgt atttttcttt tctcttctct cctcttcttt 8940 tttatttatt tatttattta tttttgagac agggtctggt tctgtcacct aggctgtagt 9000 gcagtggtga gagctctgct cactgcaatc tccgcctcct gggttcaaga gattctcgtg 9060 cctcagcttc ccaagtagct ggtgttacag gtgcatgcca ccacgcctgg ctaatttttg 9120 tatttttagt agagatgggg tttcaccacg ttgcccaggc tggtctcgaa ctcctggcct 9180 taagtgatct gcccatctct gcctcccaaa gtgctgggat tacaggcatg agccaccgtg 9240 cccggcccat atgtataata tatttcttat ccccctcccc gagcaattta gcataatctt 9300 tgaccatttt tctgtttttg tcttttaaat tagtttatct cagcaatatt aattagtact 9360 taccataatc aaaggcttga atacaatttg gaggtctatc tggagactgg acaatttatg 9420 aatagagata taagggttta gggagtggtg cctgtttaat aatgatttca ttttcttttt 9480 ttgctttctt tttttttttt gagacggagt ctcactctgt cgccaggctg gagtgcagtg 9540 gcgaaatctc agctcactgc aacctccgcc tcctgggctc aagtgattct cctgcctcag 9600 tctcctgagt agctgggact acaggcgccc gctatcacgc ccagctaatt ttttctattt 9660 ttagcagagg gggtttcacc atgttggcca agatggtctc gatctcttga cctcgtgatc 9720 cacccgcctc catctcccaa agtgctggga ttacaggtgt gagccactgc gcctggccga 9780 tttcatttta tcattcgatg cttaaatgtg atatttaaaa ttaagcattt tgtattaaat 9840 agtaatacat tcttcatgtg ctagattcaa acagtaactt tctaaagaaa aacttgattt 9900 ccctttcctc cctatctctt tgcagagctg taactgctcc tcaagtttgg tgtgtgttct 9960 acaggctttt tttttttttt ttttgagaca gggcctcact ctgcacccag gctggagtgc 10020 agccacatag tctcggctca ctgcaaccct ccatcttcag gctttcttct taacctttcc 10080 ttggctttaa atgtttttta aataaatctg tactttcaaa tagactttac tttttggaac 10140 ttttttcccc ctaagatggg ttcctgctat gttgctaccc aagctgggcg tcaaactcct 10200 tggctcaagc gatcattccc ccactgacct cccaagtagc tgggaccaca ggcacacgcc 10260 agcacaccca gctgaaacat ttttaatttt tcagaaacac tgagaagaca gtagagagtt 10320 tccatatgcc ctatgcccag ctttccctat tagcatctta tatcagtatg gtacggtttt 10380 cacaattatt gaaccaatat tagtacattg ttatcaatta aagtctgtac tttgttcaga 10440 ttttctaagt ttttacctaa tgtccttttt ctgttccagg atcccatcca ggatccagga 10500 tgatataatg tggtaatgtt acatttagtc gtcctgtctc cttaggctct tcttggctgt 10560 gagtttctca aactttcctt gtttttgatg agcttgacag ttttgaggag tattggtcag 10620 gtctttgtag aatgtccctc aattggggtt tgtttgatgt ttttctcatg attagactgg 10680 ggctgtggat ttgggggaag accacaggta aagtgctgtt ctcatcacat catatcaaga 10740 atgcacactt tcaacatgac ttatcattgt tgacattgac cttgatcacc tggctgaggt 10800 agtgtttctc aggtttctct tctgtaaaga tacgttttac cccactttcc atacttactc 10860 ttgggaagga agtcactatg catggtccac acataaggag tggggagtta tgtaacacct 10920 cgctgagggc caagtattga catgaattat ttggaatttc ctgcatgggg catttgtctc 10980 tgctctgata atttgtttag tatttatatc tgtatagact cataaatatt tattttattc 11040 ttccaattat ccaataactg ctttatttta ttgcttaaat tgcatacctc catcattgtg 11100 gatttttttt taaccacttc catacttttt ggcactaggc tcatcttata tatttcctgc 11160 actatcctag aatcaggcat ttttccaagg agcctaggtt ccttttattg aaaaataata 11220 ctacaaacca agatctgtgc tccttgctac tagggtgtca ttgcttttag gccttctaag 11280 ctgacagagc aaggaactat atatgtctgt atattaacct gtgggtatac acatctgtaa 11340 ctatttctgt atgtaccatc tatctatatt aagctaaaca taagttcata ctaatatctc 11400 cacctctaat ttgttaccat atgggtcact ctagcctact ccccttatct ataaactccc 11460 actccaacat ggagaaacct ggctcacttc caccatccat ttacatcatt cttcagttct 11520 gatatacatg tgtaagttgt atcagaatta ttaacccaca cccctgtggg aaatagtatt 11580 ataaccacta caggacaaag catatgtaca gttcctttgc ctttagtctt aaaaactcta 11640 cttatttcca aatgttactt aggtcagcac cttttcctcc acccacttga gtgaggttgt 11700 ttcaaacatt tataatacag ttatcttgtt ttgtcacatt ttgcatacat cctccagcca 11760 cctaagtgga tttttaaaat ttgcatacct tgagattctt tctttgtgct atcaagttct 11820 ataaattttc acaaatgcat agtgtctttt tttttctttt taaacaacac gttttaagaa 11880 aaatggtagg actgattttt ctttattttc caaactttct gctaatgatc atgtttgctt 11940 tctttttaaa gtacctttaa agaaaatttt atttagaaat aattttaaac ttagagaagg 12000 ttgcaagaat agtataacat aacactcata tatctattac caaattcatc ttagacttat 12060 tgttaaattt tgattaacaa tgaatacaat cctataaaaa ttgaaagcgc tacagataaa 12120 acccaagtcc ctttaatcat cacagtcctt tctctataga attcagtgtt gatttagcta 12180 cttgttgtca aattattatt attattattt ttgagacgga gtctcgctct gttgtccagg 12240 ctggagtgca gtggtgccat ctcagctcac tgcaatctct gcctcccggg ttgaagcaat 12300 tctcctgcct cagcctcccg agtagttggg attacaggca cgcaccacaa tgtccagcta 12360 attcttgtat ttttagtaga gacggggttt cactatgttg gtcaggctgg tctcaaactc 12420 ctgacctcag gcaatctgcc cacctcagcc tcccaaagtg ctgggattac aggcatgagc 12480 cactgcaccc ggcccaaact ttctttctgt ttataaatct cgtctacagc cataccactc 12540 tgaacatgcc caatctcgtc tgtttataaa tctcattttc acttgtactt ctaccagtac 12600 ctggtgccac cagttcttga gcttttgggg gatacaaaac aggttgtttt ttggctttct 12660 ggtttaggat ttagctttct tgggtctgct gagtcagtta ccacttgtca tcctgcttct 12720 agctttcaga aatttattgc tgcttccttt cctgttgctt gtaagttttt ttgcctttaa 12780 acaaacaaca ttttactcac atttatattt aagagggttt caggaggaat aagaggcaac 12840 tgtctaatct ttaattggac attatacagt aagtgtaatt ctttgtgttt catttgctaa 12900 acttgcagac ttagtgaggc caaaaacacc agccatgtca ctgttctgtc cccagacccc 12960 acatcagttg tggcctgaca catttaatga gagttgttaa attaacctag cagttgaata 13020 cttgtagaat gaaacattct gtgatgatag tgtaacagga aaggtattaa atgcttgatg 13080 cacctacttt ttggctctct ttgttcttga aggaagaggg gacctcatga ccaggcctga 13140 atcaaggtgg actctcctga atagaatacc ttttcccacc cataatgtct acctgagtat 13200 gctctactcc aggctaatga gcaagcatag cttttggaac ttctcactat cagagctgag 13260 caggctgcag tcaacatgca agcctgggtt ctaggcatgg ctctgtcact gtctttcagt 13320 gtctacattt gatttcactg tcttttcaat gtggaatatt ccattcttgg gatatactaa 13380 ttggttgttt ttagaagaaa caatattgta ggaattacat tgtgatcaag ataagttttc 13440 cttcatatat aatacaaact ttaattgaac acctgagcac ttaatgctaa gtgctgtagg 13500 ttgaatacta aatgttaaac atttctcatt tctaaagaga taaaggttgg agatgcctgt 13560 ttctcctttg tgtttctttt ctgttatttc tttctaaaat tggtaactat aaagccatac 13620 tattcttttc atttttattg tgaaattatg gatacatatg aaaggaatac atagtataga 13680 tataccttgt gcaaaataac aataataaat cagagacctg tgactgccat ccaaatcata 13740 gcaatctttt ttgaaagttt gtttgtttca cacgtattct tttagaccag tcagtaggag 13800 agagctgctt ttgccttagt tgctttgtaa ggccatgttt ctcctcaact cttccatgtt 13860 taaaatgcac tccttgtgct gagtactgat ggggatacca tacctgctgt tctcctctcc 13920 tgaaaccatc atcactgtcc tctctacagt tatagcagtc atctgtgtat ggctacatgc 13980 atagcctgtg aatttatttg gagatttgta aagtaatatg aatacttttc atagatgcga 14040 actggtaagt taaaatggta ttaagattag ctgctatttc ttcagctaag tttctgttag 14100 aaatccacct ccatgcctgt tttggccaac actcactaat gtatccatgc tgtgccactc 14160 actggtaagt gctggggtga acgcagtgat aaaccatgca aaggtctcag tccacagagg 14220 gctcacagtc tgcatcacta ttcctcgtac agtctttttc ttctcccatt tctgaattgt 14280 tagtgaagta tgatttatgt ataaaaacat gtacaatcac aaatgtgcag ctcaatgaat 14340 tttcacaaac tgaacatgca cagctaacta gtacttaaat ccagaaacag cattgccagc 14400 actccaaaag atcccttcag tgcctttcca gtcactctcc tcccgggaca tactcactgg 14460 tatgtttagt ttgcctgttc tttaaaaaaa tctttatatg aatctaatca tacagtaaat 14520 actgttttga gtctggcttc ttttgctcaa catgatgttg caaaatttac ccatgtgggt 14580 tacagtttat tcatgcacac tgctgtctca ttatgtgaaa ataccacaat ttatttacct 14640 attctactgt agataggcat ttgaataatt tccagtttta tgaatactgt tgctttttcc 14700 ctccttatgt gtcttttggt atcatatgta aatgtttctg ttggacatat aactaggagt 14760 ggaattgctg gatatgtgta aattcatttt ttgttgatat tagcaaacag ttttctaaag 14820 tagttgtaca ggtttatact cccatcagta gtgtagtgcg agaattctgg ttattatata 14880 ttctcaacaa catttggtat tttctgtttt ttattttggt catcatctgt ggttttaatt 14940 ttcatttcta taatgactaa tgaaatagag cacctttttg taagtttatt ggccatttag 15000 atatcttttt ttgtgaaatg tctattcaag tcttgtccct ttttttattg aatggcttcc 15060 ttattgattt taagaagttc tttatatatt ttggatataa gtccattgtc tgaaaaatat 15120 atcctcccgc tctttggatt ttcttttcac tttcctaatg gtgtcttctg aatgaatagt 15180 agagttctcg tgtgtgtgtt ttggttttgt tttgtgtttt aggcagagtc tcgctctgtt 15240 gcccaggctg gagtgcagtg gggtaatctc ggctcactgc aacctctacc tcccaggttg 15300 cagtgattct cgtgcctcag cctcccaagt agctgggact acaggggcat gccaccacac 15360 ccagctaatt tttctatttt tagtagagat ggggtttcac catgttggcc aggctggtct 15420 caaactcctg atctcaagtg atcgtcccac ctcggcctcc caaagtgctg gtattacagg 15480 catgagccac tgtgcctggc ccaaacaagt gatttttaaa attgttcttg aaatctaaaa 15540 gaattatttt ttttaagagg cagggtcttg ctatgttgcc caggctggtc ttgaactcct 15600 gggctcaagc agtccttcca cctcagcctc ccaaagtgct gggattacag gtgagatcca 15660 ccatgtccag ccataaaata attcttgaat agatgccagt tgtagtccat ggtcttttgt 15720 gacttttttt tctcctttgg aaggagctgc ttaatgtatc attttaaatg acaaataatt 15780 aggagcttgt ttgtgcttcc atgtttctat tttagagctt taacatatct tagatgaaat 15840 ctatatacgg ggtcttgaga ggttaaaaca gaatgtattt accacctttg catttaaatt 15900 atggactaga ttttatataa attattttaa aatgagtctg atacagccat tatttttagt 15960 attgtttact ctgtaaacaa aagatgacca gttcctgcta ttctgtcaaa gtcagggagc 16020 caggtgtgta gtctcctttt caacttgtct ttctctaggg ctttaaatcc ttctcaaatg 16080 ggccctggag ctctgagcta agcctgaatt ttcttatgga accaagaagt ggagaaaaag 16140 aactcacatt actgaggata tggactggtt ataaatagtg tagatgctac ttcttaattt 16200 aaaatgctat atgttcatga gcagctagac ttgctattaa ctatttggaa atattgaaca 16260 attgtcgctc cttatttccg tagtctttgt aagatcatga cccatcaagg acctaccgtt 16320 aacctcaaag gctcttggct gtaggttttc ctcaagctgg gtgccagaaa acagaggatt 16380 atttgcttga taatatcttg gtggagccat cagtactaga aatatttgga tgtcagacac 16440 agatcccagg ggttggcagg ttgatgataa ttttctggtg ccaccccaaa accttgcagt 16500 gctactacca taaccatggt tggctaatta gtgccttcgg ggatgagggt tgatttctgc 16560 tagttccttg cctttctctt tggatggcat gccttcactt gttacttgga cctatgtaaa 16620 gaataggaag atcagaagaa aaccataaaa ttgatgatca taggtcacag tatttgtgct 16680 ttcatttcta aggactcatt ctaacataaa taattttctc tttcaaaacc aaggtgtact 16740 catcagttat attgatacat attaacagtg atgataacca caaaaatttt aaatagaaag 16800 ctttacaaac ggttagcttt gtgaggggat ttactgcaaa attagggtat tcttttcatc 16860 tgttattgta ttgacttttt aaaacattat tctgattact tagtttttcc tttggcaaat 16920 tttgattttt aaatgtgttt tcattttgct attgagagga ttttcattta tcttttccat 16980 gggagatatt gaggatagtc taactctggt aattggtttg gtgggttttt cttttctttt 17040 ttttggctcc ctatgagcaa cttgaagatt tttttttttt tgagacggag tctcgcctct 17100 gtcgcccagg cgggagtgca gtggcgcaat ctcggctcac tgcaagctcc acctcccggg 17160 ttcacaccat tctcctgcct cagcctccca agtagctggg actacaggtg cccaccatca 17220 cgcccagcta atttttttgt atttttagta gagatggggt ttcaccttgt tagccaggat 17280 ggtctcgatc tcctaacctc gtgatctgcc tgcctcggcc tcccaaagtg ctgggattac 17340 ccacacccgg cctcttgtac tgtaatgttt ttaaggttca tccatatttc agcatgtgtc 17400 agtatctcat tttttctggg tgaataatat ttcattgtac agataagcca cattttgtcg 17460 atcccttcgt aagttgatga acattgttgg ctattatgga taatgctact gtgaagattc 17520 atgtaccagt ttttgtgtga acttgttttc agttctcttg ggtatatacc ttggagtgga 17580 attgcttgat gttaatataa atctgtgttg ctgtttttgt ttgagacagg atctcactct 17640 gttgctcaga ctgagcaaca gactagactg gactcactgc agccttgaac tccttggcta 17700 aagtgatcct cctgtctcag cgtcttgagt agctggatca caggtgtgcc gccatgcctg 17760 gctaattttt aaatttttag tagagacagg atcttgctgt gttgcccagg ctggtctcag 17820 actcctggcc tcaagtgatc ctcctgcctc agcctcccaa agtgctggga ttgcaggctg 17880 agccactgtg ttttcagcct atgtttaact tattttgagg aactgccaaa cttttccaca 17940 gcagctgtac cattttacat tcccaccagc agtgtattag agttcccgtt tctccacata 18000 ctcagcactt gttattgtct gtctttttta ttatagccat cctagtaagc atgaagtggt 18060 attgagtctt ggttttgatt tgcgttttcc tgctgactta atgatgttga gcattttgtg 18120 tctactggcc ttttgtatat cttctttgga gaaatgtctg ttcaaatcat ttgcccattt 18180 tataattggg ttgccttttt attgtttagt ctctcttatc ttgtcagaat tatttaaaat 18240 atcttcccct aattaataac aattagatag agaatttttc ttgaatgtaa ataatttggt 18300 gatgttagaa taacagagtt tatatttaca atatataaag taaatgtaca ggccacacaa 18360 acttaatgat tatttctgaa cagcagtaaa atagcatact cagctgttta ccaaaagaaa 18420 tgtgcttgtt tactttttgt aattatactg ccttgttttg gtaggattta gggactatgc 18480 agaagtagtg ggttggtaat gagagcttta aaaattgcat tttaaggtaa aagttttagc 18540 aattaactct aagatagaag tagtaaataa cattcaaaac tagagttcaa gcattgaccc 18600 agaattttct gatatttatt tttaataact taaagaaggg acttgaaact tacaagaatt 18660 ttcttgctgt ttaaatatga ccaaaccaag aaaaaattaa gtagtgaaaa tacttgcaac 18720 tttatggact tactggatac ttcagcatta tttttcacgt gctttgtttg tttgtttgta 18780 tgaattctga ggtgtatgtt gatacctttt ctcatatata aggatttatg ggttttgttt 18840 agtgtagggt tttttgttgt tctctcattc ttagtaaata tagataatcc aagaatgact 18900 tttaagtgcc tttaggtcct gccatttgct acagttataa atgttgcccc ttgcttagcc 18960 ctaatcctag ccccagctct gctattgctt aaaaacaact cattttagaa ctgcttagtg 19020 aggagggcgg tggtgaagct ttcttgctga ttgggaactc aaataatagt ttaaatctgt 19080 agggtataat gtcttaaacg tgttaaattc aagtttgttt ctattagaat tagaactata 19140 tatgatttct gtgttggaga ttcataaaaa tgtcatattt atttttaata acaataggac 19200 ctagatacag aggaaggtga taaggaggga aaaaaaaacg ctctatttta ttttatttat 19260 ttatttattt ttgagacaga atctcacttt gtcgcccagg ctgaagtgca gtggcttgat 19320 ctcagcttac tgtaacctcc acctccccgg atcaagtgat tctcgtgcct aagcctcccg 19380 agtagttggg attacaggtg catgccacca cacctggttg atttttgtat ttttagtaga 19440 gacgagattt taccatgttg gccaggctgg tctcaaactc ctgacctcaa gtgatccgcc 19500 cacctcagcc tcccaaagtg ctgggattac aggtgtgact tgagaacaaa accttagtag 19560 cacctatttg taaataaatg ttttcttaaa attgaaggtc atcaaggcca aaggcaccat 19620 ggcagattgt gacatatctg aagtaggaag agttttaggg ttcttctggg gagccattct 19680 agtctgctca gcacatagaa gggagcaaag gtaatcagga aaagagtaat ggcacactct 19740 gggtgtgaga aagtgaaagg tcatctgaat tctcaggtga ttggattatt tatttcttag 19800 cgatggaggc ataggtagtt ataggtagtt ctgtattaaa ctctactttg tatggcctgt 19860 gtaggtaatt gtatagctgt atttggaaaa aatgccattt aaatcatatt tcagccgggt 19920 atggtggctc acgcctataa tcccagcgct ttgggaggct gaggcaggcg gatcacttga 19980 ggtcaggagt ttggtaccaa cctggccaac atggcacaac cccatctcta ctaaaaatac 20040 aaaaattagc caggtatggt ggtgcatgcc tgtaatccca actacttggg aggctgaggc 20100 ggaagaatca cttgaaccca ggaggcagag gttgcagtga gccaagatgg taccactgca 20160 ctccagcctg ggtgacagag tgagattctg tctcaaaaaa ataaatcata tttgaagttt 20220 agtaaaattg gtcagaaatt atatgacagg aaaaaaaatc cagtgtccaa cagctattta 20280 ttatctatca cagtcactca aaggatatcc cccctaccct tgcccaacat ctctgtgcac 20340 tgagagcaga agttcccaga tctgtcccag ttttcagatc tgtctctctg agtatcccct 20400 acaacccaga ttctgtgaca gttaaattgg actcgggctt ccactgaccc agttgatgga 20460 agagactgag catctttcca actctaaccc aaacgtgaca atgccgcgta ttctttcaga 20520 aagtgtcagg aaagagcccg aagatccctg agctccaggg acatggcagg gaggctgcac 20580 agaagggttc ccaggggtgg aaaaggggaa tcttaaaaat aaattttgga accaagtaat 20640 caattttata gcacactcta gttctagcgc acatggttcc tccacttgtt agtgaatgcc 20700 tgacttgttg cccctgaagt gtaaaaagat gtatatttaa ggattccatt gttcagcaag 20760 ttattatgca tgcagatggc aggcactaag tgcttactct ttttgaaggt gattgcctct 20820 ccccgcgatc cccctacatt gcagtgtttt gttttaaaca gctttcttga tggagcctaa 20880 tcttatcacc agcattatag aaataagcca gagtgagaag cctgtacagt gaggtcttgg 20940 gattatacca tagtcataat ggtgttttaa attgttttta tttttcaatt gaaaaaagta 21000 cactattcta aagtagtgaa ttttagtgga gcaaactctt cacctggaag tgtttttttc 21060 cttaccaaag tgaaaattgg tggttctaag aaggctggaa tttatttgca ctgtcatctt 21120 ttactatggt gggggatgat caaatttaac atttccaagt ttccaggaga caggcagcct 21180 atcttataaa tattgtcata gaagacctct taacctcttt gttctttgga tttctgtatt 21240 tcttagaatt gtcatgcttc ctttctgaga cctaaattat gcaggggtaa cgtgattact 21300 ttgacaccaa gctcaggaaa tataaaacgg gttaagtata aaccaagcta ggtttaaatg 21360 aagttcatta ctggctacag tatattaagg gaacatattc gaaaagtctg tatttgcctt 21420 tgttcttgct gctgactctt ctgcttcttt tggtgacctt cccacagcac aagagtggtt 21480 aggtgtgagg aggctaagaa taccttgcca agagcttcaa ataatgttaa aaccagtatt 21540 ttaggtgaac catggtccca agaactctat ttaagtaatt cctagttcgg tgtcccagga 21600 gcatttggct ttactgcacc tattttggcc acatgtcttt gtttaaaagg aaaaattatc 21660 ttaggttata atacagctat tttgggagta ccttaagttt aacattagat tactaggatt 21720 cgaaaaaatg tacccagatg gtagatgctg atgagaattt gacacaagaa ttcatttgaa 21780 attgaagctt ttgtagtatt agtcagaaaa atttattgac atctggtttt gaacagcata 21840 gtgagtggag attgagatga tcagttaaaa tttaatggat tgtggccggg cacaatggct 21900 cacgcctgta attccagcac tttgggaggc taaggcaggc atatcacctg aggtcgggag 21960 tttgagacga gcctggccca cgtctactaa aaatacaaaa attagccagg tctggtggca 22020 tacgcctgta gtcccagcta cttggagtct gaggcaacag aattgcttga acctgggagg 22080 tggaggtttc agtgagccga gatcccacca ttgctctcca gcctgggcaa caaaagcgaa 22140 actacatctc aaaaaaaaaa aaaaaaaaag aaagaaagaa aatttaatgg attatggagc 22200 tctacttcac attagtacac ctcggtctct ctgtttttct gtctcaattc aagatagagt 22260 tctttgtagc ggtatggttt agaggtggta tgtattttac aagtatgtca gttggctctg 22320 tatatcctga tgataccgaa gccaggacca gacccctacg tcagttatat cactaaatat 22380 tctgttatta attttcagat tggctcctag tcccagccaa ctcatgtttt aaattaatat 22440 agttcattgt cacactggac tggaaaaacc aaatgaaatg tctttattct atgaagatga 22500 cttgtgtaga ctatacagac tagcaacagc agacactttc agtgcgagtt attttaaaaa 22560 attgaagatt tgactggcag aaaagttgct gagataggga aaagggatat ctattgacag 22620 atatatgtag aattagggag aattttatgt tttcataagt gtctcaatta tttaagattt 22680 aatttggtgt gcatgtggaa tataatggta acatggtttg tctgtgattt gtcttttgtc 22740 ttaacagatt atttttctaa ctttaaaaaa gtatacatca cttgaggcca ggagttttaa 22800 accagtctgg gcaacacagg aagaccccat ctctacaaaa cataaatttt aaaaaaagtt 22860 agccagggtc acagtgtgcc ataattgtgc cactgcactc ctgcctggac aacaagagtg 22920 agatcctgtc tctccaaaaa aaaaaaagta tgcaagtgat aaatattcac tacaaaatat 22980 ttgaacatgc agatacacaa caaaaaggaa aaattcccct gtactcctat tacctagaaa 23040 taacagtgtt gaactgtaaa tgtctcattc taggcctcct ttccattata tgtctgcctt 23100 tttaaaaagg attatatgat gcctattgtt tggcaagttt agcttttttc atgtaataat 23160 agtacactgt aaatatctat ttgagtaatg aaatttaatt tttttttttt taaagacatg 23220 gtctctctca tgccccaggc tggagggcag tggcacaatc ttagctcgct gcagccacga 23280 cctcctgggt tcaagccatc ctgctgcctc agcctcctga gtagctagaa ccacaggtgc 23340 atgccaccac tcccgggtaa tttttgtatt ttttgtagag acagggtttg ttttgccatg 23400 ttgcccaggc tagtcttgaa ctcctaaact ctcctaatct caagcgatcc cacccaccta 23460 ggcctcccag agtgctagga ttacaggtgt gagccaccat gcctggctga aatttaatgt 23520 atattttttt tgacacagag tttcactctg ttgcccaggc tggagtgcag tggcaccatc 23580 tcagctcact gcaacctccg cctcccgggt taaaacgatt cttgtgcctc agcctcctga 23640 gcagctggga ctataggagt gtgccaccat gcctggctaa tttttgtatt tttagtagag 23700 attgggtttt gccatgttgg ccaggatggc aaaatttaat ttttaatggc cgtttagtag 23760 tcctttgtgt gatgtactat aatttatttg tctcctatga ttggaattta ggttgtttct 23820 aatttttgct gacacaacac ttatatgtac ccattttgcc tctttgaata tttccataga 23880 ataacttcct gggagtagaa tacttttctt aaaggatttg tgcatttcaa ggctttcaaa 23940 atacgttgcc aaatagcttt cgagaaaggc tatttatttc agtttttggg ttcttatctt 24000 tctaattggc tttatgctta cattaaaata tgaaattttt atttagttgc agaatgaaga 24060 agagtctgga gaacctgaac aggctgcagg tgatgctcct ccaccttaca gcagcatttc 24120 tgcagagagc gcaggtaggt aacagggcaa ggtgatcacg gaatccttaa aaacactctg 24180 tgctttgaca ttacagttta aaaaaaaaaa agcttcagaa gcagttggaa gaaaggttac 24240 aaagcaagtc tggattttaa ataattgaga agtggataca tttaaaaatc acccccagtc 24300 tcttacattc atctaagaat gtcttttggt tcaattgtta tgtatgtact cttaagggaa 24360 tggctgggtt ttgtctgcta gagttagtgg aactcctttt atttcaattc agctaatcat 24420 caaaaaattg tgactgttaa aattctgcag catattttga ctacaaggat gagtctgggt 24480 ttccaaagcc cccatcttac aatgtagcta caacactgcc cagttatgat gaagcggaga 24540 ggaccaaggc tgaagctact atccctatgg ttcctgggag agtgagtata atttgccatg 24600 ttacttgatg gctgctctgt gggaattaag ttattttcat tatccaattt tgattcgtta 24660 cttatgattg tattgattag tgaaagtaga gctaatttta aaaatcaggt acatttgctt 24720 ttgggataat aaactcatct cttcttaaaa atgatgaggt aaattactag tcagacctgc 24780 tcatttttcc ttcatctctc ctattaccac tactttttca agtctcctgg gatggctgaa 24840 gtatcttgaa gcagaagcac actaatgttt ccttgattct gcacacttaa tattttgctg 24900 ttgggtggat agctgctagg gaggtctagg aagagaataa atatatgcaa atttgtagaa 24960 ttaaactcat aagagggatc ttcacattga tttagatgaa ctccgttctt ccagcagtcc 25020 agatcatgtc atttgtgggg acatagtgag ttcagattcc atctagcctg atgttgattc 25080 atggaggtgg agattgggat gccatataaa ccagtggaga ttattgggca ctcttggttc 25140 ttgatgtcag atatgtgagt gctgcatttg tcattttgta aatattcatc cagattgagg 25200 ctgatgaagg tagggcattg tgggagaatg gaagcaggat aagtccccag aaatgcgggg 25260 accatcaggc ctctaggctc tattctagca agtaactgga gtgagtgatg agcaagtaca 25320 gaagcctagt ctgggcagtg tgatggcata ggcccagatg ttcccctctt ggctttaaga 25380 tagtagacag tttgcagacc acacctactg tgaggagcga agctcctaaa atcattgctt 25440 ggtagaaata gtttatgagc ccaatctagg atgatcctaa atggcgatga tgtgcattca 25500 gttcttacag gtaccaggga gaaactaaaa accctagtgt ctaagggcaa gaccatcata 25560 atgtaactgc cgtatattgg aagcaaacca catgcttggt actgtgcttg atgcttttat 25620 atattatgta tattgtttcc aatcttcata gtaaccttga aaggtagaga ttatattatc 25680 tccattttac agatgtgaaa atttaggtcc agagagaaaa agtaattttc ccaaagtcat 25740 atgactgata agtagcagaa gtgaaagtca aacccagata ttctagaatc caaagtcctt 25800 gtttgtttca ctgtactctg cagatctgtt gcaatatatg gtaaatagat cagtatggaa 25860 attagctctg attaatcaca taactcttca gaatactttc atttgataca agtacatatt 25920 taggaacttc ttaaagggct ctagtattca gcagacatgt ataaagcact ttttaaatat 25980 aagatgcagt atattttaca gtggataggt aaaaagcttt gtataaggca gtgttcctca 26040 ttgttattca aaagcctaaa atccaactag ggtaaagggc ccacaaatac tgataactaa 26100 gacacataag agcagtgggc atcaagaact gaaacttttt actgggtgat cataggggaa 26160 aatgagattt gatgtgggcc cggagttttg atgggtaaga acattccaga gggaaaaagg 26220 cacagaaagc aggtgtgaat tcagccaggt gaaggcagtt ctgtggatgt agtaattggc 26280 tttgtatgag caagagtttg tgaagggaga tagaaagtcc tgactacaac tgttgtttat 26340 agaatctttg gaatgttaga tgggcgtatt taaacttcat atgtagcttt tagagaatcc 26400 ttcagagttt ttgagcaggg gtatgagagg acttgtgtgg tgttttggaa acattaactt 26460 ggcagcaatt tctaggatgc atcatggtag aagagatacc aagaggacag gaagaacagt 26520 tagaaggtgt tcgtaatatt attagatgtt acttccatat attttttgga gtatatcttt 26580 gaatagttat gcaaaaaaca attagctttt aattttggca aaaccttatt ctgctgatac 26640 gaagtaagaa tactaggtat ttaatgtttt aaaaaattaa ttataaaaat tcgtttttat 26700 agaaatttca aacaatagag gaagtatgtt aagttagaaa aggaagctgc cattgccacc 26760 gacttccctt tctctagttc ccactcccca ggggaaccac tgttaacaat ttgggtcttc 26820 cagacccttt tctgtgtaca acaagttttg atattcccta aaatattttt catttaagga 26880 gccctcattg aactgtgtac aaatctatca cttttaagta tatattcaat tatttttaaa 26940 tatctcaaca cacacctata tcagttaaaa agatagccca catgtgttgg tattgttcat 27000 gatcatgtta tttctagttt tgcaatatta cgttatcaag ttaggatgga aaagatctta 27060 tctgctgata aggttagaag tgagatttag atctgtatac caatacaaat acatgcttat 27120 ggagttgagc agtgggcagt tctgtggact attcatgtat gatcgtacta ttcttttttc 27180 ccctatatca gcctcgtaaa gcacttgagc tggttgattt tatctttttt tttttgagac 27240 ggagtctcac tctgttgccc aggctggagt gcagtggtgg gatcttggct cactgcaagc 27300 tctgcctccc gggttcatgc cattctcctg ccttagcctc ccgagtagct gggactacag 27360 gtgcccgcca ccactcccgg ctaatttttt tgtattttta gtagagacgg gtttcaccat 27420 gttagccagg atggtctcga tctcctgacc tcgtgatctg cctgccttgg cctcccaaag 27480 tgctgggatt acaggcatga gccactgcac ctggctgatt ttatctctaa ttactaatat 27540 tttaggtcct aaacaggtcc tcgattactg atattctaga atttaaacca aatgtgtaag 27600 taaattttgt cttttgtctt ttcttgaggt atattttttt aaaggaagag taaatagttc 27660 tttgtgaact attcttgaag tgtattgttt ttgaagtgta ttaaattttt gaagtgtatt 27720 gttaaagaac ttgggttata agatgaaaat ttcatttaga acactgggga tagaagaaag 27780 atttgcttct tttctaatta tatattaata atgcaaaatg gaattttaaa cacacattga 27840 gcctttaatg ctcataattt tgaactgaac caaagcaata acttttttcc ttgctactca 27900 aatttaaccc ttcatttttc tttatgtctt ccctttgcct agaaataatt ctttttcttt 27960 tcatttagga tgaggatttt gtgggtcggg atgattttga tgatgctgac cagctgagga 28020 taggaaatga tgggattttc atgttaactt ttttcagtaa gtatgtatgc atatcagaac 28080 caccccctac aaactagagt gctttggatt atgggtgaat ctgactgaat taaaataact 28140 agttgagcta atatattaat acattatgtt tatttggtgc cttttactgg aatattcaaa 28200 gcttaatata cctttgtttt aatatccctt ctctttgcct tctattttca tgaatatctg 28260 tggaaaatat aattaaagca gttatgagga tgctgcattt ttacatgagc taagcctcat 28320 cttccaggtc tacggggccc ttttgcgttg cttctactca tacttaatct gtagaatagg 28380 aattaaatgc ctttgatgaa ttttgctctg aaagcttctt ttttctgaca gatcaaagtc 28440 agcctgattt tctcccttca tgttacttgc catttatctt tttgtgtgcc tcacatagaa 28500 gtgagcaacc atttccttta tttgttgtat tagaaagata agaaaatgca atgcttttag 28560 aggtgatgtt atataagttg tcaaaattca gagaacagga ggatgagtga cttgaatcag 28620 attatttggg ggcacttatt taatgttgaa ggcatttatt ttatttactg gactatatat 28680 atataacaca cacagtaaaa agcagaggtc aggctgggca tggtggctca tgcctataat 28740 cccagcactt tgggaggccg aggcaggcag atgccctgag gtcaggagtt cgagaccagc 28800 ctggccacca tggtgaaacc ccatctctac taaaaataca aaaattagct gggtgtggtg 28860 gcaggcacct gtaatcccag ctactcagga ggctgaggca ggagaatcga ttgaacccgg 28920 gaggcagagg ttgccgtgag gtgagatcgc accattgcac tctagcctgg gggacaagag 28980 tgagacttcg tctcaaaaaa aaaaaaaaaa aaagaggtca aaggctgggc acagtggctc 29040 atgcctgtaa tcccagcact ttggaaggct gaggtgggcg aatcacctgg ggctagaggt 29100 tcaagaccaa cctggccaac atggagaaac cccatctcta ctaaaaacaa aaattagccg 29160 ttagccgggc ctggcggtgc acgcctatag tcccagccac tcgggaggct gaggcaggag 29220 aatcacttga acctgggagg cagagattgc agtgagccaa ggtcgtgcta ctgcactcca 29280 gcctgggtga cagagtgaga ctcagtctca aaaaaaaaaa aaaaaaaaaa agcagaggtt 29340 gtaagtatac agtttggtga atgttaaggt ttgctttttg ctgttttgtg ttttttttgc 29400 tttttccttt tgagacaagt cttgctctgt tgcccaggct gaatttggag tgcagtggtg 29460 tgatcacttc tcagtgtacc cttgacttcc tgggctcaag tgatcccccc acctcagcct 29520 cccaagtagc tgggacttgc aggtgtgcac cactgtgccc ggcttttgtt tgtttgtttg 29580 tttgttttgt ttttttcagt ttttgtagag atgggtctca ctgtggtgct caggctggtc 29640 tcaaactgct gggctcggga tcctccaacg ttggcctccc aaagtgctag gattacagat 29700 gtgagctatt gtgctcagcc tattaaggcg tttttgaaag tgactagcag tagacataag 29760 tgtgtaaaac ctgataggga ggagtttttc ttgtttgctt tttggggaaa gacagaagaa 29820 tggatgtttt agaggaggta tggtgacggt tgtggaggac aggctgtctt ttatcctttg 29880 gaaagtcatt taaacagagg caggtgtaag gcactcttgt tcttgggtaa caggacatgt 29940 ctaacatctt tcccctcctc tgcttttgca gtggcattcc tctttaactg gattgggttt 30000 ttcctgtctt tttgcctgac cacttcagct gcaggaaggt atggggccat ttcaggattt 30060 ggtctctctc taattaaatg gatcctgatt gtcagggtaa gttgtataac agaaaagatg 30120 ggctctacag agaggcaata atcagaatat ttttgaatat tcgatttttc atttgctttt 30180 ttaaaagtta tttcagtgtg ttaaatgatt tttggtttgt ctgtggtttc atgatgaagc 30240 tgaaccaaaa tcatttgcaa atggttcatg cacacaagtt ttgttgtcaa atctattgaa 30300 aactggtcta tcattaatat tgttcaccat ctgctttgtt tgtttttaat gtttataata 30360 tgataatgta tatgttctga aattagttgt ttttataata aaaggaagga tattacccct 30420 ttgatatctc atcaaaagca gccattcctc tcattaattc tgttacagtt ttaatctagc 30480 tctcttcttc cttcttttct tttctccttc tcttcccctc tcccttccag gattgcttat 30540 gccaaaatgt ttggggagcc aaaactgttt aacaagtatt gctccattag gagcctacct 30600 ctcaagtaaa ttacagagat gtatgtcttt agtgacttca gagttaccag ttagttaaat 30660 ttgcatgaag taatttacag ttgtaaaaga tctaggcttt taaagtatcc tctgatttca 30720 gataaattta ttgctatcgt caaccttata aacaaaacca atttatccgt cataaattgg 30780 catacttaac taactaatgg gtgttgcttg accactgtag tttttctacg tagtgccacc 30840 ttatttatta taaaacattt gttaacgttt ttagggttcc agaatccttt gggaatctga 30900 tgaaagttgt aagcttttcc cccacatagc taatatgtac tcatctataa taatggactt 30960 taggttaaga actcgagccc taaagaggtt ttcattcctt ccctaaattt taattgttac 31020 tttattaaca aagaaaatac tatattttac ttttcgaact acattaaact cttggacacc 31080 aaagcctcca aggaaaagag aaataaaatt aattccagtc tttttttttt ttcaaactaa 31140 attttcaact gcatttaaaa agtgaatcaa atgggccggg tgcggtggct cacgcctgta 31200 atcccagcac tttgggaggc cgaggcggac agatcacgaa gtcaggagat cgagaccatc 31260 ctggctaaca tggtgaaacc ccgtctctac taaaaaaata caaaaaaaat agccagacat 31320 ggtagcgggg gcctgtagtc ccagctactc gggaggctga ggaaggagaa tggcgtgaac 31380 ccaggaggtg gagcttacag tgagccgaga tcgtgccact gcactccagc ctgggcgaca 31440 gagcgagact ccttctcaaa aaaaaaaaaa aagttaaatg aattaacttg tatcaggtag 31500 gatgcttttg actgtgagta acaaaaacct agctcaaaat ggcttagaaa aaaattttta 31560 attgtttttt taaacgggga aattttattt cccctgaaaa caagtccctg acaaggttag 31620 ttaagtcagt ggttctttga catcatcaag gacagctgac atatcagcat attggcacag 31680 gtcccccctc ccctcagggt taaaagatgg cagtttcacg tatcagtatt acattgaaac 31740 acgacagtgc tcaagagaag aaaaagaact atttaaaaaa aaattttcta ccacctttag 31800 aaagaactgt ttttttcttt gagaagcaag gaaagcttct cagaagccct caatttccac 31860 tggtcagaat ggcatcctgt gcccattcct aaaccagcaa gggaagggga ttaccctggt 31920 atacatgata aagcttccaa agtcaagaca aggctggagg agtaggaaga aaatcaggcc 31980 tcattagaaa gaaggcaatg gtatcatcta ctcaggaggc tgaggtgaga ggattgcttg 32040 agcccaggag ttcaaatgca gcctggacaa catagtgaga tcctgtctct taaaattaat 32100 aataataata attgcttttg gcactgaata ggcaaagtaa aacgatcatt attgaaacac 32160 atttctagaa gagcaaatat tatgtgggga aaatgttttc taaaaaaacc aatttttgta 32220 tactttcaaa ataaggagtt aataaagcta aaagctgtca tatttaacaa ttgcttttcc 32280 ttgaaaggaa atagatttgc atttattgta accatttcaa gctagtaata tgtaaaatac 32340 tttcacttat attttaaaat tattctaaag gctaagtgat ggcattggta gaacaaataa 32400 gtcttgctgg tcacggtagc tcacgcctgt aatcccagca ctttgggagg ctgatgtggg 32460 tggatcacct gaggtaggga gtttgagacc ggcttgacca acatggagaa accccatctc 32520 tactaaaaat ataaagttag ccaggcatgg tggtatatgc ctgtaatccc agctactcgg 32580 gaggctgagg caggagaatc gcttgaacct gagaggcaga ggttgcagtg agccgagatc 32640 acgccattgc acttcagcct gggcaacaag agcgaaactc cgtctcaaaa aaaaaaaaaa 32700 aaagaataag tcttgcattt tgtgattttg tgtaccctta agttaaaatg agaaattgtg 32760 tctgttaagc tgctataaag tgttttgcct tattttttag ttttccacct atttccctgg 32820 atattttgat ggtcagtact ggctctggtg ggtgttcctt gttttaggta agtgttttta 32880 atgtaagaaa aggcaagaaa acattacatt aaattttata agtaaaatta cttgtagtaa 32940 atgttcatct tgattactgt atagtaatat attaacaatt aataataaac atttttaaaa 33000 tgttgctttc ttaaaaatag tctttcgtta ccatcagagt aacatgcacg ttaataaaaa 33060 gtttctttgt ggttgtaggt tcttcctaac ctatacatat attcttctcc cccatgtgat 33120 aaatggggta tgctgatgac tctgtagtct gtcagttttt ttccccttgt gagctattgc 33180 cacacagcat tcacagactc aaaccagaat tgcttatacg cttactgcag ccagttgaac 33240 taactcataa gtgcaggtgg cccatctttg gtgggtctgt cagtagaaaa cagaaaatat 33300 gtaacctcgc tagacataac ttctgaaaga accaccattg gatttccaga atgctttagt 33360 ttaccgttgg tgagaataag aactatttta caataggcaa gaggactttt tttttttttt 33420 tttttttttt ttttacagag tcttgctctg tcgcacaggc tggagtgcag tggcgtgatc 33480 tcggctcact gcaagctcca cctcccgggt tcacaccatt ctcctgcctc agcctcccga 33540 gtagctggga ctacaggcac ctgccaccac gcccggctaa tttttttgta tttttagtag 33600 agacggggtt ttaccgtgtt ggccaggatg gtctcgatct cctgactttg tgatccgccc 33660 gcctcggcct cccaaagtgc tgggattaca ggcctgagcc accatgcctg gcctggatta 33720 tattttttac tctaaatatt tatttttaaa tacttttttc ctagacatta aaacaaagag 33780 atgaacattg gagtaaatta tttcaatgtt ggcttcttga ttatatgtaa agaaaatata 33840 tatgatacag ttagagagac caccatcact tctgtcattt ctacaaaaga ataacagggc 33900 catcttgtgg ctattaaaga tggtgtgatt gcctcactgt gcttctgtga catcagtttc 33960 tcctatgtcc tgttttgctt cttgttgccc ccttaattaa gatccttggt ggtggttctg 34020 ctaatggttc tggctaggac atcctggcac ccatgcctgg caggtgttta ataaattgag 34080 gacctgggcc aggcatggtg gctcacgcct gtaatcccag cattttggga gactgagtca 34140 ggaggatttc accttgaggc caggatttta agaccagcct gggcaacata gcaagacctc 34200 gtctctacaa aaaaattttt ttaaattaac caagcatggt ggtttgtgcc tgtagtccta 34260 gctacttggg agactgagcc aggaggaccc cttgagccca ggagttggag cctgcagtga 34320 actatgatgg cgtcactgca cttcagcttg ggtgacagca agacagtgtc tctaaaaaca 34380 aaacaaaaca gttcaggacc tgttttgcca aaactgacag agccttgcat tgctgaccct 34440 acattttact tagaaaacat aaatcattca tcgcaccatc taccacctcc tcaacagcat 34500 catgtactct atgattggtc attagaattc taaggaaaga gaaatagtaa ttttttttcc 34560 tctattgatt attgagaact tcagtaataa aaatgaccaa caggagaata atttagaaga 34620 aatagttcct tggcttgctt attttctaaa ctgtaccttt aagagaaagg ctgaccaccc 34680 gtgtttcccc aagtcaaatc ctgaatggac caaagaggag cccttaagcg tatgttcaga 34740 ttgattgctt atggcataaa taatttaaag aaatttaatg gattgattcc cttttaaagt 34800 ttcatgggag gaggttttat aatccttcat cattgatcat tcttgctatg taccatttgc 34860 ttgtaagcaa aaaataagag tttggcccgg cgtggtggct catgcctgta atcccagcac 34920 tttgggaggc cgaggcaggc agataacttg aggtcaggag ttcgagacca gcatggccac 34980 cgtggtgaaa ccccgtctct actaaaaata caaaaattag ctggctgtgg ttgctcatgc 35040 ctatgatccc agctacttgg gaggagaatg gcttgaaccc gagagacgga ggttgcggtg 35100 agctgagatc gtgccactgc actccagcct gggtaacaga gcaagattcc atctcaaaaa 35160 aaaaaaaaaa aaaaaaaaaa aaatatatat atatatatat atatatatat taataagagt 35220 ttggatacac ttatgagcat ttgtgcccaa tattcgggtc attttgggaa cttccctttt 35280 gaagagaacc agtttaatgg tgttttgtct tgcagagctc tgtgcagagc tcccaagctg 35340 gctgctcttg agctatagtt agcctatagt ttatttgttc cacagttatt tttcaagttt 35400 ttttaaatta gttgccaaca ttttaaaaat caaaattatt tcacataaaa cgcctgaatg 35460 tttttacttt tcttgaaagt tggaatatca agaaaagcta gcctacattt cacatggcac 35520 cctttggctg gagctgagtt gttgctgtct cctccataca ggtcatgtgc cttccagttc 35580 agtaagatcc caccaccata ccccaactta gtatttgaag gggtaacccc tgctcaatag 35640 tcacttgttg ctttgggtcc caaacagaat gatgttcccc aagctcagag ctgacagctc 35700 ttggttactg ctaaaaatca gttcaactgt ccaagtacca ttttctacca ctgagtatcc 35760 cagtaaaagt atttaaaaaa aaaaatgtgc taaaggctca gagaccatct ctgaaagagg 35820 aaggccagga ggagtatctg agtttccact ggaggggcat atcacctctc tgctgacagt 35880 tttaggaggt acaaactttt gtgaatgtat agattctggt gtagtcacac aaaaagagtc 35940 ttactaagaa tagagaaata ggctgggtac agtggttgac acctataatc ccaactcttt 36000 gggaggctga gacaggagaa tcacttgagc ctaggagttg aaggccagcc tgagcaacat 36060 agcgagaccc ctatctctac agaaaattaa aaaattagct gggcatgaac ctgttatctt 36120 agctacttgg gaagcagagg caggaggatc atctgggccc aggaattcaa gcttgcagag 36180 gctgtgatca taccgctgca ctccagcctg ggcaacagag caagaccctg tctgttaata 36240 ataataataa taataataat aataataata gtagacaaag tttcaagttg agacgatgag 36300 aattgctgta atagataagg ggatcctgac atctagtgga caacattata tactgtcgac 36360 agaaaaaaca gcccatgaca aggaaggtta cattttttct ttttacttga gaaaaacaca 36420 gttataacct ctttttgttg ttgttgttgt tgttgttgtt tggagttcat tatcttggaa 36480 tgcttttttg gaactcaata ggtagatttt gatgtttctc aatgaggctt tcatatgtaa 36540 atgtttggct ccatcttatt ttttatttcc tttttagacc aattcaattc tgaatgtgca 36600 aaagacacta ttgactctta cattatacct tctcattgaa ggatggataa tttggtatga 36660 aaatcctctg cagattctgg aggaaattgc taatgttttg attaccttta gcagaaaaat 36720 aacaatgtat cgctatcagg agagggattt ctggggaggg gacttaacat ggaaattata 36780 aattcattca tgacttttct tttttaaaat taggctttct cttgtttctc agaggattta 36840 tcaattatgc aaaagttcgg aaaatgccag aaactttctc aaaatctcct caggaccaga 36900 gttctcttta tttattaaag gtattaaaga aaaaatttat tctagtccca gtgtaaccat 36960 gtgctacatt gtagatttct gttcagtttt agagtaagta tctgtgatag gggcatatag 37020 agaaagtaaa gaaggcacag tccctgcctt ttagcagttt cttgtctagt aacagggatt 37080 tttacaaaaa gctattgata aagtcaaatt agaaaaactg ccataggaga aatcattact 37140 tcatgctttg acagtgtgaa agaggcaggg gtcttagcag tcatcagaaa gctgggatat 37200 ttggctgctt gactgaattc cagcatcctg aagccactgc ttactgttgc accagttcca 37260 gaggatgaca ttcagaagga acccttcagg cctaatcact catctctgcc cctaaatccc 37320 agaacctgga gagactgtgt agtactttaa gaactccttt tgtctgggct gctgacaagc 37380 ccaccaagtc ttccatcttt taaaattgtc attgaggctt tccccatggc tttcttcagg 37440 ttgacattct gataccactc taatgtgagg aaggtcaaac tcaagaaaag aggagaaagg 37500 accttttccc ttgcctagaa caggaggact aagccctgcc aaggctcttt gattcacaca 37560 tgattttccc cagaggctga cactccatct tacatcaatg gctgcctgaa ggaagaggta 37620 gagaatcttg agaaagagta caccagagag agctctctgt ggtagcaagt tctaataggc 37680 tttcttggac ttggctgttt gttccttggg gctttgggga ctgtgttaat catagagtgg 37740 aaccttgagc accacctaga ggccagtcca gttgaaggtc ttaggcaaag cattataaag 37800 gataggtggt gtagttcaga taaactttac atgatagtgg agagtgtggg gaagaatttc 37860 agaatcatct gaggcagagt gaagagcaaa aatagggaag tatggggctt gttcagagga 37920 tagcagcttg attggggcat aaaggagtat tgagggtcaa ggctaaaaag tcaggttgaa 37980 acaagtttct cagatacctt gagcaccaag cccatttgca gtagaaccat gaaattgata 38040 gagccagcga catgttgggt caatgcaaga ctaagcccac gtggagcacg aatttgttct 38100 gtactcgcag agtgtcagtt tatataggca atcatatact aatcgtgagg ttctcaattt 38160 tgaagacttg tggtggtctc aggaggtatg cttcaagaaa ataaagcatc taccatagtt 38220 gattttccag gtgatagaac agcattgaaa gcttttaggc aacagaatca tataatgaga 38280 ccctcttgaa tataacaaac attttttcaa gcatagtata gtaaactaaa attaacactc 38340 atggagtttt ccatgtgtca gataatatat taagcatttt acatggataa ttaaatgtat 38400 gtaggtaaag aaggcttaaa ctgcttttaa agttagtttc attattaaaa aaactagtat 38460 tcattattaa aaaacaggaa tctttgattg catgtctgaa aatgtgtagc atgtcatgaa 38520 tttgaattca gtcacacagt ctggacacag atggatattc ataccactgg cctgggcctt 38580 aatgcctaca atgtgtcttt gtttatgtct agaacataag agacttatta gatattcttt 38640 gcagttttat ttagttacca ttgtcatctt tcatctatat atcagattgg aatttatagt 38700 tagccttagg tttaactttt acctatcaag gattccaaag ctagtactgt tgggttgcta 38760 caagtcctcc ttatgaaggg tgctgaaatc acctaatatt ttccctctaa tacattgaca 38820 cccaagatat actgagggga cagatttctt tctcttttta aaaaaagtca tcaattctgt 38880 aggtgtagtc taatgttagc tctgaatttc taaatggact gagatcacac ctctgtctat 38940 tctctttgtt cttcagttag aaagaaatct gaatctttat taggggattg cataccttct 39000 tcatcttggt tctaagcata agaaatagca gcccagttga cagaaagcct gaacgtctca 39060 gttttcctgt gttgtttggt gctttactga aaattttgtt gttattgttt ggattttcta 39120 aaatttctga aatgtttttt gatattctaa atttaaaact atgtatgtgg ggttgtgatt 39180 tgaagtaggc agtccttagt aataataatg ttgtaggaag tactaaggga gattcaagtg 39240 ttgagcctgc tggtgcttca ggagtgtacg ttaagagttg tcagaaactt tctcagaggt 39300 aaatgaatgc acaccattgg agggcataat agagtttatt tgcttttcta tttaactgtg 39360 ataccctggt ttatttgaaa tcattcctga cacacccaga gaattcagag ctctaaatta 39420 agggtttgag gaacttacag cagaatttag aattctaatg acttcactaa ttttccaggg 39480 atccccagag ggaacctgaa aagccataat atcctatggg actgagttcc ctttgtccac 39540 tgaaagggta agaactctgt ccagatccag atgtagaatc ccttctttct ctagttcaga 39600 ggacatttag tttaattttg ttagaagaca tactagaaaa ccgtcatgtg aagtaagaaa 39660 gctttatttt tttcctccct atcgattctt tattatgctt atcacagttt ttaaaaagta 39720 attatctgct aattccaaca tcaggttaga taagtctatt tgtattgatg gatttttctc 39780 ttgactctgg gttacatttt cctgttttta cacacctggc cagttatttc tttgttgcag 39840 cattgttaaa ttatcatgtt gtagagatta tgagctctga attttgtcag aaggttgttg 39900 agtttttgcc tagcaggcag tcaaattact aaaggataaa tttgatctta tttctaactg 39960 tcatgtacag tgattcttct agggagatag ggatagggga gagggaaaat gtagtttaaa 40020 aatcctaggc taactcctgc tgaggaaaat cactggtaaa ctagtgacta ctctgctaaa 40080 ttgcctctct tgacattgtc aacctgaagc catcaaaagg atcagaatac agagtttatg 40140 caagtagaaa gctgggaatg gccaatcaga gaacacaaac tttagaaaat ggggtcagtg 40200 ttcccaaaag ctttattttc ttttatggac ttctacttct ttgatggaga tgtcaccaat 40260 taaaaggtag ctgaagaaag acacaggtag ggaattttta aaataacttt gccagaaaga 40320 tacagataca gaatttttaa aataacttta ccaaaacaga aaaataatat actttccata 40380 ttgtactaag atttggcatt ttactagaaa agggcataag aatagtgact tttctagtca 40440 ggctacctga aatatagtac aaataaacca atcttgaagt gatcccaaaa cacctccttc 40500 taatcagtca aagtagatat tacaaaagct aaacattaaa gctcatgtac tactatcata 40560 ggggcagctc tcaggttcct tttcagagtc catttaagct taagggaggg aaaaatgtat 40620 taataagctg ttatctgttt tcctacttca taacaaaatg ccagaaggaa ctgctaaagg 40680 tgtgcacagt ggccatcaag aagtatgtga ctgggccggg cgcagtggct cacgcctgta 40740 atcccagcac tttgggaggc cgaggtgggc aggtcacctg aggttagaag tttgagacca 40800 gcctgggcaa catgacaaaa ccccgtctct actaaagata caaaaattat ctgggtgtgg 40860 tggtgtgcac ctgtaatccc agctacttgg gaggctgagg cagaagaatc gcttgaaccc 40920 aggaggtgga ggttgcagtg agccaagatc acaccactgc actccagcct gggcgacaga 40980 gcgagactct gtctcaaaaa aaaaaaaaaa aaaaaaaaaa aagtatgtga ctgaggaagc 41040 agaagaaaca gatacctgtt ataaacatct gtacctgaaa tggtaaaaaa tgtaaagtgt 41100 ttatactttt cttatttacc agttcactgg aaaatacatt tttatggatt accacctcct 41160 ctgatgctgc caggtgaatg aggagttagc tacagtaata aggtggtaac acagttgatt 41220 ggttatatct aacactattt gtgtggtacc aaaagcactt gtacatatga agcataatat 41280 attttaaaat tatttatttt aactgcatct gaaatagaag gaaaaaagga ggtttttgag 41340 ccctagcatt aatgagctag ggccttcata attaaaaaag tgacctagtg agctgtacct 41400 attaggcatc aaatatttta atttttttcc atgtgataac agacactttc atttctcctt 41460 ttctgtaatt tttctcctgt tccttaatac ttcttttatt ttattaaagg aaattcatct 41520 caaggtaaag gtttacttat tggactatgt tataagatag caagataata gggaaatggt 41580 gtttccagta gaatttttgc taaacaaaat ttacatgtct ccaagaagaa ctggttgtct 41640 tcctcttgga attaaatgat ctgtaggcta ttggattcct tttttttttt tttttttttt 41700 acaaagtgat ctgatctata atttagtaat acggtccaga gtccctaaga tctgtttgtt 41760 cttcagtttg tgtactgcca gcatttctgt aacactgcca ctcaagcctc caagtctcag 41820 acagcagtgc cctctagcag tctttcattt ggagattgtg tggtgattga gattacccac 41880 tggtgacctc tctaggtcct cttgagatat tttattttaa tttaattttt acttctctta 41940 ctgaacaagc aaagggattt ttaatgtttg ttgggtgtca ggctttgact ttgagggatc 42000 tattaaagct gtttacctat gctaaagcat cataaactaa ggaagtggcc aggtaattgt 42060 ccgtaccata ttgtatattc ttgcaaactc tgttttgacc tactagtcac tgtttcatca 42120 gttttcatct attaaaagaa actgggccag gcatggtggc tcacgcctgt aatcccagca 42180 ctttgggaag ccaaggcggg cagatcacga ggtcaggaga tcaagaccat cctggctaac 42240 acagtgaaac cccgtctcta ctaaaaatac aaaaaatcag ctgggtgtgg tggcgggcgc 42300 ctgtagtccc agctactcgg gaggctgagg caggagaaca gcatgaaccc ggggggcgga 42360 gcttgcagtg agccgaaatt gtgccactgc actccagcct gggtgataga gcgggactcc 42420 gtctcaaaaa aaaaaaaaaa aaaaatatat agaaactgat aaaacaaatt atttatgttt 42480 ataatgagtc tggcctcatt gcaaaaacat tcatttaata ttcatctgcc aattttcttt 42540 tttgcttctg cttggctttt agagttcctc atatttttgt gtctctgtct ctgtgctttc 42600 cgaggtcttt caatctctta tgtctctcaa tctaaagaaa agatgtcctt gtatcagttt 42660 cagtggttta cttcaaatca caacacaaat gaatttatag tttggaaaaa tgttgataat 42720 attaaaatat gttcatttcc tccagtgtgt ttgtagtctc tctccatttt ggcagccttt 42780 taaatactta cgcagtaatc taatcagaaa agatacagaa gacaccgggc atagtggtgc 42840 acgcctgtta gtgccagtca cttgcggggc tgaggcagaa ggttggcttg agccctggag 42900 gtgtaggttg cagtgagccg agctcgcgcc actgcactcc agcctgagtg acagactctt 42960 gtctccaaaa aaaaaaaaaa gaaaagaaaa gaaagaaaag acatagaaga gtttcttgtg 43020 gcttttatag taatttggag gatagaaaag taagttatag gccgatattt gagcccatcc 43080 aactctcact tcttgctgag taccacaaaa tagtttctca ataaatgttt attaagcagt 43140 aaatgtatct ctttagtgtt tcttctttta ttcttaatag ttcttctttt attgttaata 43200 tagggaatta cttgtagttt ttcccaagtt attttcatga gaaccaaaac aatatactag 43260 tacagaaatg cctcaaagta ttagatgaaa actgcattta gtaaaatgcg taggcattat 43320 ttcgagagca tttatttcag tcagatatat tcctttctta aaatttagtt tgaaagtaca 43380 tgtattattt taagagaaac atgccccaaa tacaatgact ttttaggaaa acttgttttt 43440 taaaagtatt tgtggtacct gtgagtttat ccttgttttc taattttgcc atgctcggtt 43500 gaggtatagc atgacatggt tggcatcctt actggaaagc ttccttgtgc aatatctagt 43560 ggaattgtta gggtaaacag tatcttcagg tatattagga aaattacttg tgcacttgga 43620 ttatgctctc tgtcccaagt ttcagtcatt gctgacagcg ttcagtgtgg gatgcgcagt 43680 agcactgctt ccattgtatt cttttgttaa gaggtttgga aagagagact cctgacttca 43740 ggagttttta gaaaaagcat tccagttttt tctagttcta gaggccttga taactttaag 43800 aactgaaatt ttaatgttta ttgaataccg tgatgtcagt atacagtaat cttaaaacta 43860 tgttaaaatg cacttgtgtc tccaaacatc ctgccttctt ttgtgactac atccttctct 43920 tagccaaaat aacaagctaa ctagagtttc aatattcaat atccttctct ggcagatgtt 43980 ttctggcaaa ggccttcctg catttatgaa ttctctctca agaagcaaga gaacacctgc 44040 aggaagtgaa tcaagatgca gaacacagag gaataatcac ctgctttaaa aaaataaagt 44100 actgttgaaa agatcatttc tctctatttg ttcctaggtg taaaatttta atagttaatg 44160 cagaattctg taatcattga atcattagtg gttaatgttt gaaaaagctc ttgcaatcaa 44220 gtctgtgatg tattaataat gccttatata ttgtttgtag tcattttaag tagcatgagc 44280 catgtccctg tagtcggtag ggggcagtct tgctttattc atcctccatc tcaaaatgaa 44340 cttggaatta aatattgtaa gatatgtata atgctggcca ttttaaaggg gttttctcaa 44400 aagttaaact tttgctatga ctgtgttttt gcacataatc catatttgct gttcaagtta 44460 atctagaaat ttattcaatt ctgtatgaac acctggaagc aaaatcatag tgcaaaaata 44520 catttaaggt gtggtcaaaa ataagtcttt aattggtaaa taataagcat taatttttta 44580 tagcctgtat tcacaattct gcggtacctt attgtaccta agggattcta aaggtgttgt 44640 cactgtataa aacagaaagc actaggatac aaatgaagct taattactaa aatgtaattc 44700 ttgacactct ttctataatt agcgttcttc acccccaccc ccacccccac cccccttatt 44760 ttccttttgt ctcctggtga ttaggccaaa gtctgggagt aaggagagga ttaggtactt 44820 aggagcaaag aaagaagtag cttggaactt ttgagatgat ccctaacata ctgtactact 44880 tgcttttaca atgtgttagc agaaaccagt gggttataat gtagaatgat gtgctttctg 44940 cccaagtggt aattcatctt ggtttgctat gttaaaactg taaatacaac agaacattaa 45000 taaatatctc ttgtgtagca ccttttactg tagattagtg cttaatttct tggcttgcat 45060 ttgttgattg ctaaggcaat tttttctaat cttagggaat cattcagtag atgcgattaa 45120 aaaactaatg ttgggtcaat ttttttcttc attttcagca caagaagtcc tcttatatcc 45180 tactaaatac attcctaaaa atgtatttga acattggttc tgtaaaagat aatggactaa 45240 aaaagtagag aggagttgta gagatcttaa atcattctgg aattcctaat tatgcttcaa 45300 tttttagaca taattttaga taatttattt ccagtgtttt ctgcatgttc tcatttgttc 45360 tttttctcag ttgaatgcac caactggttt gagtcctgtg agcattcagt cagttgaaat 45420 taaagattcc tcatttctcc tgatttctat tcttgtctca atcttaaatt tagagaccag 45480 ttgtttttat gatatcagcc atttgatttt tttcattttc tatttaagaa atatgaagaa 45540 aaaatacacc aagatggtca aattactaca caaatcagca ccagcacagt ctgatagctg 45600 caaatgtcca ttcatctgct gtgtatgtat atccagaatc agcataggaa gtcgttcagg 45660 atatcagtat ataatgcaca gaagtgtggg ttgtttgaaa gccaaacagg aaaattagga 45720 gcctcctgga ttgacatttc aatgatccct ctaaccagtt tatggattat tatgaataat 45780 agtgtagtgt gttctttttc agaagttata tttgataata gagaagggag ttttatggaa 45840 gtttctttga agattttttt ttttccattt cgaatcagat tatagcaaca atggagtttg 45900 gaagtttgta tggcctataa tgttctaagt tccagaatga aaagatctgt aacaatctga 45960 atagatgtgg acacatatag cagagagaac tatgtaaatt atcttgcaga acaaaataga 46020 agggtcctaa atcacgttaa ctcaaacatt gtagactagc tttgtgttta ttcttcaggt 46080 ccttgcgcct tatttggttt tgtatattca acgaactgaa atatttggaa ttcctatttc 46140 tacgtatttg gtggtccata agactttgtc aaatgtaaac ctacagtttg atacgcttta 46200 aaatacctag ttaagaggat gatttctctt taatcgttta aatgttctga aaattaaaat 46260 cttttgaggc acatgaagtg ggcaccatat atcatctaga gtccttactg gtattcagga 46320 tgaaaatgtt cacgctgcat taattgtcat ttttctctcc catgttcttt ctcactttga 46380 tacgttaata ctgataatgg ataaagagtg agtttttata ataaatggtt ttggaaaggt 46440 attcatagga accgcggtta tttacttaag gttatggagt aaactagctt ggaccttggg 46500 ctgcaggacg actaggattc acccataacg acacagtgcc ctatgtttct taacttcttg 46560 ttgccatttg aaactctgta ctcttatgtt taaagggttc tgtatagcca tttttttttt 46620 cagaaagtta cattgctttg tatagaaata aaaggcatta ttaaaatttg cttgttaaaa 46680 aatgataaga atgaatatct ttacttatag ggtcaatttt gtttagagtt ttaaagaaac 46740 aacagaagtt ttagtgaagt aaatgatttt ttttttccct ttgctttctt attacatact 46800 gacttgagct ctggcccacc gtgagtcatt attagtacag atgaaatttt gtttggtctt 46860 cactatgtgt tttttgtttg tttgtttttt gagacggagt ctcgccctgt cgcccaggct 46920 ggaatgcagt gttgcgatct ccactcactg caacctccgc ctcccaggtt caagcgattc 46980 tcctgcccca gcctcccaag tagctgggat tacaggcgcg cgccactacg cccagctaat 47040 ttttatattt ttagtagaga cggggtttca ctatgttggt caggctggtc tcgagctctt 47100 gacctcgtga tccgcccgcc tcggcctccc aaagtgctgg gattacaggt gtgagccacc 47160 gcacccagcc ttcactgtgt tcttatacta aaatatactc tacttagcta taaatggttt 47220 acattcaaag gaagaagtaa gataactact gaagtagagt atcaggacca atagaagtga 47280 tatttcaatt tgagtgattg cctgaggggg taagattctg gggtgagaat ctagacccta 47340 acaaaactag acccctggcc tcaggcttta catccagtcc aatgccctct tcccattgtc 47400 ccaaccttct ggagggttgg cgtgtaaaca ttgacataaa atgaaaaaaa tcatttgacc 47460 gtgcttcttg taaacatcag taatctaatt ttaactgaaa acatgctaag aatagacatg 47520 gaattgtaca tttatgtaaa gtaggaggat gtgtgtttgt atagatagat ggatgcatag 47580 attttttttt tttttttttt tttttttttg agacagggtc tcgctctgtt gcccagactt 47640 aagtacaaag tcgtgatctt ggctcactgc aaccttcacc ttccaggctc aagccatccc 47700 acctcagcct ccccaagtag ctgggactac aggcatgtgc caccacaccc agttaatttt 47760 tgtattttta gtagagacag ggttttgcca tgttgcccag actggtcttg aattcctggg 47820 ctcaagcaat ctgaagtgct ggaattacag gcgtgagcca cctcacctgg ccagtaggag 47880 ggtatatttt atgaaaaagt agttgtgacc cctattacct atgtgaatct ggttcttggt 47940 taaacctcag tggtgaatat aggttaatca tgtgcaaaat gtataataat tttcaggttc 48000 tgtgggtggt tcagctgttc agaagatttt cacctctcag gcttttagaa tatgagttga 48060 ggaatgatga gggtcataac agaaggcaaa atatgcttcc atagatggtt tatatattta 48120 aaaaaaatgt ttttttgcgt attttaaacc ctaaatagtg tttctatcct gtaagatgga 48180 taaggtggat tgggtgagag catttgttaa agcagtagta cagtactctt gcatctcctt 48240 ttcccttttg cgtatacctg tacacgtttg tgtatgtgta taattgtgtt cttttagact 48300 acctacagaa agtgcccttt gacaagtagc ctctgcatgt tggttctttg taggctctac 48360 tgttaatgtg gaagacatct caaagaaata cactgggaat tcccaaaggg gagagtaagt 48420 ttaggttgaa agtgaaagag gtgttcagga ggaaatgatg gttgtagaaa ttgatttttg 48480 tatcatctcc attctaaaaa gaagctattt tataacccag ttgattgtat cacctacaac 48540 tttatggcct atagaaagaa ctgctcttac gtaaagtaca tgaattgaga gctgtcctcc 48600 cgaagccagt ttccctgact ataaaatctg aaatagtcca tctactgcgt ggcattttaa 48660 tcccctcagt cctccattat tctcagaatt ttccgcttat aggataaata cattactcaa 48720 aggttttgtg ttaattttta tacctgtttt tacttatagc ctgtttcttt ttttctttac 48780 caaaaagcat cgtcttctga ttttgagagt tttaattctt aagtatcagt tgatgaatta 48840 ataaaatgta aatgtaaatg cacatctctt aaacctgaat acagaacata ttaatataga 48900 ggattctcct acatttatgc aggattttac ctaggtcaga aactgagatg caattgtgtg 48960 aaatagatgt cgtccaacct gctttgtatg ttgttcattc tgctctaaaa acagctaatt 49020 ttaatcattg atgtaggaaa taaatgtttc acagactttt ccccattcat tagtgattgc 49080 agattcatag acatcaaaac tgagaaaagt ggtttcctct aagccttaac aaagattagt 49140 gctggagctc tgggaagatg gcagtattca ttgttattct tcagttgtct ggaaggttcg 49200 cgagtgatat acgcagcctg ggaaacaaga aaacaggtca aaaatctaaa agaaatctct 49260 aaaatagaga agatgatttc cctgacaaag cattattaga aaaagtataa aactaaggaa 49320 gttctgcctt ctggttatga aacccagcct gattctaatg aaattcaacc aaaatgccaa 49380 agctagagtg attttcacct tgcatagttg ggtttgcatt cgtgtcccct gctgagagct 49440 gctttgcctc ccacttcccc tcccccattt aagccagata cttcctgagc caagttggag 49500 aggactggtg caacctatca tcataactgg gctagagtta ctcttgatga aacatccctc 49560 ttttcatttg cagaactgtc agaaatgcct aaaatgctgc ttaagtgagg ttttagaaag 49620 aattttgcca tctgcttcct gattccacat ctttattttg acagaacatt tcataggccg 49680 tatttggcag ttcctctgtt tggatgtcat tttaatatgt tgcttttttc tctgattatc 49740 aacactgagg attttcatgt atatgattta tttccagttt atattttaac agattgcaga 49800 taaatcagtg attgcagaga aatcagatgg atcaacattt tcactattta ttacttttta 49860 tgtatttatt actatttatt tattactagt atgctttggt actatatagt tgatgaaaat 49920 agtgggcact ggtgacctga agaagcaaaa ttagcagtcc atatttttat aggtttgatt 49980 cagtgactct caagccctag 50000 7 6564 DNA Homo sapiens 7 atctgtatat gcatggtcat gtgtaggaca aaaatcatct ttttttctct ttttggttaa 60 ttatatattt tgtccctgag actgtgaagt gagttgaatc tgtataacct atttttgtcc 120 gtaagctctt ctctggttta gcagttctct agatttaggt attggactta tatatatatt 180 tttccaatat ccacctcatt gaattataga cttagttcct acagtagttc cacagagctt 240 ccaaataagg gtaaggataa tgggtaagta agtacttacc tctcatcttc acttccatag 300 aagaaggctg agtgcagtac atttgtatct acactttata aattatgaac tagtttttca 360 tcaaaaaatt tcatggatgc caggcacggt ggatctcgcc tgtaatccca atactttggg 420 aggccaaggt gggcagatca cctgaggtca ggagttcgag accagcctgg ccaacatggt 480 gaaacctctg ctctactaaa aatacaaaaa ttagccgggt gtggtggtgc gtgcctgtaa 540 tccccagcta cttgggaggc cgaggcagga gaatcacttg aactcagagg cggaggttgc 600 agtgagccga gattgtgcca ctccagcctg gacaacagag tgaaactcca tctttaaaaa 660 aaaaaaaaaa aaaaaattga tggttgggta gattaagaat tattttgtct ttatataggt 720 ctcaaagctc atggccacaa tattttcctc ttcagaatgg caggactatg ggaaggaatg 780 tgcattcaga acaacgggga aaggctatag ggaaattaat ttcaaaatat ttggctgagt 840 gattgtggac tcttccttgg aagtctttaa acacagaata gatcctccta tttggaatga 900 cttaagggta aaggaaaagg aatggattag caggcgcctt aaagacccca agaactttag 960 agatttgtat gtggcccatg tcagtgacag atggaacagt aagttcaata gacgtcatgt 1020 ctgccgctcc caggctatgc ttcctttaat gtagtgacct tctgggagca ttgtcctcaa 1080 ttgtctgcaa gagggaaagg agtacatcta aatttctggt aaacttaatt tgggtggctt 1140 attacctact gttatgtatc ttttaaagat ccagtgatcc aaaaagctgt gctttgccca 1200 aaattccaaa atgagaaaac cataaaaacc accctctagt ttctggagcc attttcaaat 1260 ctgtgttgat tctgaaggaa tttcatttac gtagcatttt cacatgttaa cagtccttcc 1320 cctaagggat tatgggtaga ggaaagaaga tgaggtggta aggaggcact ggaggagatg 1380 aagtctgatt tgtttagaaa ggaccgatgg aggagtgtag cagacactcc ttttgttctt 1440 tgctcctgct ttaatccttc agacccacag gacaagattg ccagctaatt gcaaggagcc 1500 cgtaaaagag aattggccta aattgaagaa taaatatatt ttttttttaa ttcttccttt 1560 atttgggtca ctttgcttta catatgttta tgtctccaaa aagtggttgg ataccctctt 1620 gagaggtttt ctttacaaaa aagaaagaaa aaaaaatctg aaattccaaa gcctctttcc 1680 ctgccattgt agcacttagg caaaagcatg aagaactaaa tccactagac tgtagtttaa 1740 acttgaaaaa acaaaaacaa aaaaccccaa aaagactata atgtgcttta gagtggcatt 1800 tttatacaag tgactcattg ctagcagtat attgaccatt ttaaagtgca atctgatctt 1860 tcatgggtag acatttaaat tctctgagta cagacactgt acttgggaca aattattata 1920 tttgtacctt ggtgccaatc ttaatcatgt actcattggg tgcttatttt caaatacctg 1980 atccttctaa aaacagtata ctaaaacctg ttgttaaaca tatatcaagt ttaatagcaa 2040 gccatggata ctttttactg ccttttattt attgctgggt gtattttatt agaaggaaag 2100 gtaagtaaat gtgttttgca gaaaatatgt agtataagtc agctccagta gcagttatta 2160 ttttttaact agatggcgga gcatttacac acttagttta ttagtgacct tcccataaaa 2220 tttttaggaa cacagggcca aacagaggtc ataagtttat ccaagccaac ccacttcttt 2280 ggtgacatca gacaactgaa gagaagccaa gaaaacgttc cctccatctt caaattctat 2340 gtgtccttgg ctttgcctcc atccatagtg gtttattccc ctcagaaaga attgcagatg 2400 gaatttgtat aatcacctgt gttagaagtg atgatctact ttgaaaaata cagtttttat 2460 gtattataaa taattgtgct ctgccttctt gcatatagtt gctttgggcc tttatttgat 2520 aaaaactatc ttattagcca gttatgacat acatgacttt atgctggatg tagtatatac 2580 aaagatttgt acaagacagc ccctgaacac aaggaggttt tagtctggga ggagattggt 2640 aggagacaaa gtgctttcct gtccaagaaa tcttaatatg taatgttttt ctgcttcgtt 2700 tgcatagtga tggaagaagc tttttacact tttcaagctt gctgctaact taattttcac 2760 cagaagggca cttttaaaga tgttcgtaaa accaaatgcc ctcaaaggag agattactgc 2820 cacttgaaga ggccaccccc aaaagggcca aaaggatggg ggaacatctc agggtgctat 2880 gtggccagga cctagaaaac cagctgccaa ggggacttgt gactgttagc ctagggcttg 2940 agccctcgct ccccatttac tggctgtgta atcttgggtc atttgcatgt cctatccaag 3000 ctttggtttt ctaatctgtg tgctggaata ttttctcaca gggccatttt tagggaggta 3060 aagtgcccag catctagcaa acagctcttc acagctgatc agctatagta ttgggtctaa 3120 gggcaggaca tgaaaagccc aaacctataa ttcaaaccga gggaattcat ttccttttca 3180 ggaccgtata gctctcataa accctgcctt tcctcattcc ttctttccta aatgtcatta 3240 ggccatccct gataaatcag atcattctta tcctcctaaa ttcctgcagc ccagatgaga 3300 atatgattta ctattgcttt tctggtaata ttatcatatg tttatgtggg aacttatata 3360 catgttcatt tgatcttcac agattactat aaaagggatt attttgcgtt ctttaccacc 3420 aagcaaccaa agcctccctc ccccggcaaa taattttcca acactcacta acatcaaaac 3480 actctacaga ttcaataaac tttgattctc atttcctttc ctgagttcta taggctgaaa 3540 ctctggcctc ttttttgctt agtccatttg tattacatta caagacttgc cgtatactat 3600 ctttgtatgg atttaggtat tcgaagcagc ctttgaactt cttatccttt gctttgtttc 3660 tttctgcact ggccttacat tctctgctct tttccctgcc ctttccatgt cttctttgcc 3720 gccctctcga cacctccccc ttcccctacg tttccagtgc tgcttagatc actcttgtta 3780 cttctctgtc ctaaattcct acacaactgt agatagggcc attttctcag tggccactct 3840 ccctttcctt tctccaaatc ttaaccctgg gccatttgaa ccctagttac ttcttttttt 3900 tgttttgttt tggttttttg gtttgtttgt tttgagacga agtctcactc ttgtccccag 3960 gctggagtgc aatggcgtga tcttgactca ctgcaacctc cgactcctgg gttcacgtgg 4020 ttctcctgcc tcagcctccc gagtagctgg gattacaggc gcctgccacc gtgcccggct 4080 aacttttgta tttttagtag agatgggggt ttcaccatgt tggtgaggct ggtctcgaac 4140 tcctgacctc aggtgatctg cccgccttgg cctcccaaag tgctaggatt acaggcgtga 4200 gctaccgcgc ccggcctaac cctagttgct ttctatcttc ccattccatc tagccgcatc 4260 caacaagaaa accacatgcc tgctttctct tcctgaagct ctccaaggaa cgttcctgta 4320 tgcccctgga tagatggttc cagcctgtgt ctgtgccccc caacccagtc ctgccaatcc 4380 aagcctgccc agatagggcc tgcctgtttt cccctttatt gggggatctg gggagctttt 4440 cacagtggtg gcgtttgtca cagtgttcac aggctcacct cttcatgccc cttcagtact 4500 agtttggctt ggccattaaa cagaaagcat tggggtgtcc tctgctcttt gttctctctc 4560 actatataga tcacctccaa tggcatctgc atgaatctat gacaactctc gggcctccat 4620 ggagaagcac ctggctagtc ccaaatttca taatccttct gatgcttctg aattaccttc 4680 cacaaaacac taggattcta taaactttta attggaattt tatcttttaa aaacagcctt 4740 aatcatgtaa taagatgtat tttcaaatat gtgttcaatt gtgtggttat ttgtgatctc 4800 atttacagac atgaacaaat actaaaacaa tatataaata aatttactca gagtatttgg 4860 gatctcagat tttgttttac aaaaagggtt ctgcagctga aaaaaaaatg gcttgaaaac 4920 cactgcccta agtaaacact catctcttac tttgcctaaa actgttagac cgttatacat 4980 tttttttcct ccgtcaatct tacaggggtt tcaaaatgcc ctgattttgc aaaacttatg 5040 agcacatcta aaaagtcact ttctaagcct actatctcca aaaatcaact gtttgttctt 5100 tttcagactt gtttttgtga atttattatt tttacatagg tagtactagc atactggcac 5160 agcccagtga gatgaggtct cttaagccat tctgtttata tcagatgtta aattttttaa 5220 atagtctttg ctgcagtgta gattatcttg tctttttctg cttttcagct cgttctgaca 5280 aatgccccat gatgtttttg tttttgtttt ttgagactga gtttcactct tgttgcccag 5340 gctggaatgc aatggtgcct tctcggctca ctgcaacctc cacttccctg gttcaagtga 5400 ttcttctgcc tcagtctccc gtgtagctgg gattataggc gcccgtcacc acactcggct 5460 aatttttata tgtttagtag agacggagct tcaccatttt ggccaggttg gtctcgaact 5520 cctgacctca agcgatccgc cctcctcggc ctccccaagt gctgggatta caggtgtgag 5580 ccaccacaac tggcctcgat gatttttttt tttttttttt ttgagaagga gtttcgctct 5640 gtcgtccagg ctggagtgca gtggcgcaat ctcggctcac tgcaagctcc gcctccggag 5700 ttaacgccat tctcctgcct cagcctcctg agtagctggg actacaggcg cccgctacca 5760 cactcggcta attttttgta tttttagtag agacggggtt tcaccgtggt ctcaatctcc 5820 tgacctcgtg atccgcccgc ctcggcgtcc caaagtgcta ggattacagg cttgagccac 5880 cgcgcccagc cgatgatggt gtttttaaag ccaacttcta atacctcttt tttccattgc 5940 ttcattgcca agaggaataa ttagactgtt gcaacaatta tgacaaaggc gtcctatacc 6000 cttggtcttc tttcatccat agggcatcca aaagagacct ggtttctctg ttgattggaa 6060 gttcacatca aattccttga ccctgggatt aaacctacag taaacatgtg tatttttgtg 6120 ttgcagatgc gtatcataga tggatagtaa tgaataccat ttattgagca tttatcatgt 6180 gcgaagtact ctgctttctt cttcacaggt agatttaatc atctccccaa ccctttgagg 6240 tagggaatac tgtgatcctt gttgtatatt tttaaaaaaa tggagactca tgttaactga 6300 cttgcccctt accgtcctta agtggggagg ctgggattaa tttggtttgt gttgttagag 6360 catgaactct ttaacaacaa tagaacactc atcatctttc ctgtgtgtgc acctgcttcc 6420 atttcttttt attctcctgt cccttcttga tctacacttt aacacattcc tttctcttcc 6480 cacaggattc cctttattga aggtgggaat gagggggatt ctggactgca ggcacaggac 6540 atctgccctc ccacagcagc ctga 6564 8 24 DNA Homo sapiens 8 agaacacaga ggaataatca cctg 24 9 22 DNA Homo sapiens 9 tgcactatga ttttgcttcc ag 22 10 25 DNA Homo sapiens 10 cattttaagt agcatgagcc atgtc 25 11 23 DNA Homo sapiens 11 cttgccttag gcttatctcc ctt 23 12 22 DNA Homo sapiens 12 gcctcggaat gtcagctact tt 22 13 19 DNA Homo sapiens 13 ggtcatctgg tgcctttgg 19 14 22 DNA Homo sapiens 14 ccagcctaac aatgctctcc tt 22 15 21 DNA Homo sapiens 15 cggtgcttac agcctgagaa g 21 16 16 DNA Homo sapiens 16 aacgccaacg ccatgg 16 17 25 DNA Homo sapiens 17 ttaatgtttg aaaaagctct tgcaa 25 18 26 DNA Homo sapiens 18 tgacaacacc tttagaatcc cttagg 26 19 25 DNA Homo sapiens 19 cttcacccct tagtagctct gtgtc 25 20 24 DNA Homo sapiens 20 aatccttgag aactgcttgg atct 24 21 20 DNA Homo sapiens 21 ccctttgcac ctgttcctgt 20 22 17 DNA Homo sapiens 22 tccggtctgc caccatg 17 23 31 DNA Homo sapiens 23 aaatgtcaga tagcaagtat tatagggttt g 31 24 20 DNA Homo sapiens 24 tgccaagcac tgagcttcac 20 25 19 DNA Homo sapiens 25 aaccacgatt ctgctgcca 19 26 15 DNA Homo sapiens 26 gccccacccc cgact 15 27 19 DNA Homo sapiens 27 acacttgagc ctcggaggc 19 28 19 DNA Homo sapiens 28 cagggaatca gagcgagcg 19 29 16 DNA Homo sapiens 29 cccgcctctt ccccag 16 30 14 DNA Homo sapiens 30 agccccggcg tcga 14 31 17 DNA Homo sapiens 31 tgtcgactgc gccatgg 17 32 15 DNA Homo sapiens 32 ccgcctgtgg gtcgc 15 33 18 DNA Homo sapiens 33 gcctcaggcc tctctggc 18 34 19 DNA Homo sapiens 34 gagctatcgg cagcagcag 19 35 19 DNA Homo sapiens 35 tccctgagtc agaacccgc 19 36 18 DNA Homo sapiens 36 aatacacccg gcacacgc 18 37 17 DNA Homo sapiens 37 cccctcgcct ccgtttt 17 38 31 DNA Homo sapiens 38 ccaccattac tgtatgttag attaacactt c 31 39 27 DNA Homo sapiens 39 tcattttctc gggtttgatt taataca 27 40 17 DNA Homo sapiens 40 aaaccagtaa ccggccg 17 41 29 DNA Homo sapiens 41 tccatagaat aacttcctgg gagtagaat 29 42 25 DNA Homo sapiens 42 ttgtaacctt tcttccaact gcttc 25 43 31 DNA Homo sapiens 43 tggttcaatt gttatgtatg tactcttaag g 31 44 33 DNA Homo sapiens 44 aaaattagct ctactttcac taatcaatac aat 33 45 29 DNA Homo sapiens 45 gaattttaaa cacacattga gcctttaat 29 46 26 DNA Homo sapiens 46 ccagtaaaag gcaccaaata aacata 26 47 21 DNA Homo sapiens 47 ggcaggtgta aggcactctt g 21 48 20 DNA Homo sapiens 48 tgtgcatgaa ccatttgcaa 20 49 25 DNA Homo sapiens 49 gcattttgtg attttgtgta ccctt 25 50 26 DNA Homo sapiens 50 ttaggaagaa cctacaacca caaaga 26 51 26 DNA Homo sapiens 51 cagattctgg aggaaattgc taatgt 26 52 26 DNA Homo sapiens 52 gggactgtgc cttctttact ttctct 26 53 31 DNA Homo sapiens 53 ttctcttagc caaaataaca agctaactag a 31 54 26 DNA Homo sapiens 54 ggacatggct catgctactt aaaatg 26 55 30 DNA Homo sapiens 55 gatcatttct ctctatttgt tcctaggtgt 30 56 36 DNA Homo sapiens 56 catacagaat tgaataaatt tctagattaa cttgaa 36 57 22 DNA Homo sapiens 57 catgtccctg tagtcggtag gg 22 58 31 DNA Homo sapiens 58 gttttataca gtgacaacac ctttagaatc c 31 59 25 DNA Homo sapiens 59 aacacctgga agcaaaatca tagtg 25 60 27 DNA Homo sapiens 60 ggatcatctc aaaagttcca agctact 27 61 32 DNA Homo sapiens 61 aattcttgac actctttcta taattagcgt tc 32 62 24 DNA Homo sapiens 62 atcaacaaat gcaagccaag aaat 24 63 24 DNA Homo sapiens 63 aatgtgttag cagaaaccag tggg 24 64 32 DNA Homo sapiens 64 ctctacaact cctctctact tttttagtcc at 32 65 29 DNA Homo sapiens 65 ttgtatggcc tataatgttc taagttcca 29 66 24 DNA Homo sapiens 66 ccacttcatg tgcctcaaaa gatt 24 67 27 DNA Homo sapiens 67 cgccttattt ggttttgtat attcaac 27 68 23 DNA Homo sapiens 68 cgcggttcct atgaatacct ttc 23 69 27 DNA Homo sapiens 69 gagtccttac tggtattcag gatgaaa 27 70 30 DNA Homo sapiens 70 caagcaaatt ttaataatgc cttttatttc 30 71 28 DNA Homo sapiens 71 gttatggagt aaactagctt ggaccttg 28 72 29 DNA Homo sapiens 72 ttcatctgta ctaataatga ctcacggtg 29 73 19 DNA Homo sapiens 73 gctggaatgc agtgttgcg 19 74 27 DNA Homo sapiens 74 ttgaaatatc acttctattg gtcctga 27 75 17 DNA Homo sapiens 75 gacctcgtga tccgccc 17 76 23 DNA Homo sapiens 76 acaagaagca cggtcaaatg att 23 77 22 DNA Homo sapiens 77 aatttgagtg attgcctgag gg 22 78 27 DNA Homo sapiens 78 gccaagatca cgactttgta cttaagt 27 79 23 DNA Homo sapiens 79 acaaagtcgt gatcttggct caa 23 80 21 DNA Homo sapiens 80 gctgaaccac ccacagaacc t 21 81 27 DNA Homo sapiens 81 gttgtgaccc ctattaccta tgtgaat 27 82 25 DNA Homo sapiens 82 cacaaacgtg tacaggtata cgcaa 25 83 21 DNA Homo sapiens 83 ggtggattgg gtgagagcat t 21 84 29 DNA Homo sapiens 84 ctataggcca taaagttgta ggtgataca 29 

What is claimed is:
 1. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:4 or a functionally equivalent variant of said amino acid sequence.
 2. An isolated polynucleotide according to claim 1 which is cDNA comprising the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or a DNA comprising a nucleotide sequence which hybridises to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 under stringent conditions.
 3. An isolated polynucleotide according to claim 1 which is a genomic DNA comprising the nucleotide sequence of SEQ ID NO:6 or SEQ ID NO:7 or a DNA comprising a nucleotide sequence which hybridises to SEQ ID NO:6 or SEQ ID NO:7 under stringent conditions.
 4. An isolated polynucleotide comprising a consecutive 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair portion of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7.
 5. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:4 or a functionally equivalent variant thereof.
 6. An isolated polypeptide comprising a consecutive 10 amino acid portion identical in sequence to a consecutive 10 amino acid portion of SEQ ID NO:4.
 7. A method of producing a polypeptide according to claim 5 which comprises culturing a host cell containing an expression vector containing a polynucleotide sequence as specified in claim 1, under conditions suitable for expression of the polypeptide and recovering the polypeptide from the host cell culture.
 8. An isolated polypeptide encoded by a variant of the nucleotide sequence specified in claim 1 having a sequence polymorphism correlated with a disease.
 9. An antibody which is immunoreactive with a polypeptide comprising SEQ ID NO:4 or a polypeptide according to claim
 8. 10. An antisense oligonucleotide comprising a nucleotide sequence complementary to that of a polynucleotide encoding a polypeptide according to claim 5 or a variant thereof having a polymorphism correlated with a disease.
 11. A polynucleotide probe comprising at least 15 contiguous nucleotides of a polynucleotide according to claim 1, or a complement thereof.
 12. A pharmaceutical composition comprising a polypeptide (A) comprising the amino acid sequence of SEQ ID NO:4 or a functionally equivalent variant thereof, a polynucleotide comprising a nucleotide sequence encoding said polypeptide (A), an antibody which is immunoreactive with said polypeptide (A) or a polypeptide according to claim 8 or an antisense oligonucleotide comprising a nucleotide sequence complementary to that of said polynucleotide or a variant thereof having a polymorphism correlated with a disease, optionally together with a pharmaceutically acceptable carrier.
 13. A method of treating an inflammatory or obstructive airways disease which comprises administering to a subject in need thereof an effective amount of a polypeptide (A) comprising the amino acid sequence of SEQ ID NO:4 or a functionally equivalent variant thereof, a polynucleotide comprising a nucleotide sequence encoding said polypeptide (A), an antibody which is immunoreactive with said polypeptide (A) or a polypeptide according to claim 8, or an antisense oligonucleotide comprising a nucleotide sequence complementary to that of said polynucleotide or a variant thereof having a polymorphism correlated with a disease.
 14. A method of detecting genetic abnormality in a subject which comprises incubating a genetic sample from the subject with a polynucleotide probe according to claim 11, under conditions where the probe hybridises to complementary polynucleotide sequence, to produce a first reaction product, and comparing the first reaction product to a control reaction product obtained with a normal genetic sample, where a difference between the first reaction product and the control reaction product indicates a genetic abnormality in the subject or a predisposition to developing a disease.
 15. A method of detecting the presence of a polynucleotide according to claim 1 in a cell or tissue which comprises contacting DNA from the cell or tissue with a polynucleotide probe comprising at least 15 contiguous nucleotides of a polynucleotide according to claim 1 under conditions where the probe is specifically hybridizable with a polynucleotide according to claim 1, and detecting whether hybridization occurs.
 16. A method of detecting an abnormality in the nucleotide sequence of a polynucleotide according to claim 1 in a patient which comprises amplifying a target nucleotide sequence, in DNA isolated from the patient, by a polymerase chain reaction using a pair of primers which target the sequence to be amplified and analysing the amplified sequence to determine any polymorphism present therein.
 17. A pair of oligonucleotides useful as primers for amplification of a fragment of a polynucleotide according to claim 1, each oligonucleotide of said pair being at least 15 nucleotides in length and said pair having sequences such that when used in a polymerase chain reaction with human genomic DNA or a suitable human cDNA target, they result in synthesis of a DNA fragment containing part or all of the nucleotide sequence of a polynucleotide according to claim
 1. 18. A variant of a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising amino acid sequence SEQ ID NO:4, which variant contains a sequence polymorphism correlated with asthma.
 19. A method of determining predisposition of a patient to asthma which comprises identifying in DNA from the patient a sequence polymorphism or haplotype in a nucleotide sequence encoding a polypeptide comprising amino acid SEQ ID NO:4, as compared with a normal control DNA from a non-asthmatic subject, which correlates with asthma.
 20. A method of identifying a substance which modulates the activity of a polypeptide comprising amino acid sequence SEQ ID NO:4 or a functionally equivalent variant thereof or a polypeptide according to claim 8 comprising combining a candidate substance with said polypeptide and measuring the effect of the candidate substance on said activity. 